Ochlerotatus mosquito species collected around Finland in 2015 and 2017 were studied using next-generation sequencing. Mosquito homogenates were sequenced from 91 pools comprising 16–60 morphologically identified adult females of Oc. cantans, Oc. caspius, Oc. communis, Oc. diantaeus, Oc. excrucians, Oc. hexodontus, Oc. intrudens, Oc. pullatus and Oc. punctor/punctodes. In total 514 viral Reverse dependent RNA polymerase (RdRp) sequences of 159 virus species were recovered, belonging to 25 families or equivalent rank, as follows: Aliusviridae, Aspiviridae, Botybirnavirus, Chrysoviridae, Chuviridae, Endornaviridae, Flaviviridae, Iflaviridae, Negevirus, Partitiviridae, Permutotetraviridae, Phasmaviridae, Phenuiviridae, Picornaviridae, Qinviridae, Quenyavirus, Rhabdoviridae, Sedoreoviridae, Solemoviridae, Spinareoviridae, Togaviridae, Totiviridae, Virgaviridae, Xinmoviridae and Yueviridae. Of these, 147 are tentatively novel viruses. One sequence of Sindbis virus, which causes Pogosta disease in humans, was detected from Oc. communis from Pohjois-Karjala. This study greatly increases the number of mosquito-associated viruses known from Finland and presents the northern-most mosquito-associated viruses in Europe to date.RNA viromes of nine commonly encountered  Sindbis virus (Togaviridae: Alphavirus) [Peribunyaviridae: Orthobunyavirus) [Negevirus was isolated from mosquitoes collected in eastern Finland [Mosquitoes  are vectors of a variety of medically significant pathogens worldwide. The known endemic mosquito-borne viral pathogens in Finland are havirus) ,2, Inkoohavirus) ,4 and Chyavirus) ,6. Threeyavirus) , Hanko vyavirus)  and Ilomyavirus) . Of thes Finland .Aedes, Aedimorphus, Culex, Culiseta, Dahliana and Ochlerotatus [Ochlerotatus are most numerous, with 23 recorded from across Finland, but distributions vary according to species-specific life strategies. Halophilic species, including Oc. caspius and Oc. dorsalis, are usually restricted to costal locations, while other species, including Oc. communis and Oc. punctor, are widely distributed across the entire country. In Lapland, the dominant human-biting species include Oc. communis, Oc. excrucians, Oc. hexodontus, Oc. impiger, Oc. nigripes, Oc. pullatus and Oc. punctor/punctodes. Further south, the most commonly encountered human-biting species include Oc. cantans, Oc. caspius, Oc. communis, Oc. diantaeus, Oc. intrudens, Oc. pullatus and Oc. punctor/punctodes. Mosquitoes, generally, are abundant in summer months between June and August, but only Ochlerotatus species have been associated with the known mosquito-borne pathogens in Finland, although vector species associations are not yet confirmed. Sindbis virus has been isolated from mosquitoes twice: once from a pool of unidentified specimens at least containing Ochlerotatus [Ochlerotatus [Ochlerotatus species. Inkoo virus was first identified from Oc. communis and/or Oc. punctor/punctodes from Inkoo in southern Finland [Ochlerotatus or Aedes [Forty-three species of mosquitoes are recorded from Finland, which belong to erotatus . Some sperotatus ,12. Spec Finland , while Cor Aedes .Ochlerotatus is a widely distributed genus, with 199 species located in tropical, subtropical and temperate regions, and is known to include several species which are naturally infected with arboviruses [Ochlerotatus, Oc. caspius, Oc. communis, Oc. dorsalis and Oc. excrucians, are classed as being of particular interest for targeted surveillance due to their vector potential for a series of listed pathogens by the European Centre for Disease Prevention and Control [Culex pipiens, it is of interest to first pursue the study of identified Ochlerotatus species in order to ascertain their potential virus associations, particularly when they have been implicated as vectors for all three endemic mosquito-borne viruses in Finland and are regularly attracted to humans. As such, females of nine commonly encountered species, Oc. cantans, Oc. caspius, Oc. communis, Oc. diantaeus, Oc. excrucians, Oc. hexodontus, Oc. intrudens, Oc. pullatus and Oc. punctor/punctodes were chosen from suitable specimens that were collected for a nation-wide distribution study [Ochlerotatus mosquitoes that were collected from a variety of habitats around Finland in summer 2015 and 2017, 514 unique sequences of RNA-dependent RNA polymerase (RdRp) > 1000 nt, belonging to 159 viruses, were recovered. Of these, 147 potentially novel viruses were identified as well as sequences belonging to 12 established viruses, including Sindbis virus. Final decisions on the taxonomic placement and species’ status of these viruses will be determined by the ICTV.More broadly, oviruses . In Euro Control . Althougon study  for inclOchlerotatus. A subset of 2333 of these deep-frozen adult female specimens was chosen for inclusion in this study (see below). Notes were made if any specimens were visibly engorged with blood, or if they had ectoparasites (Acarid mites).As part of a larger study, 52,466 mosquitoes were collected from around Finland between 2012 and 2018, using a variety of collection methods and from a multitude of different habitats . The priOchlerotatus, by species, collection location and collection date (Oc. cantans (n = 1), Oc. caspius (n = 11), Oc. communis (n = 35), Oc. diantaeus (n = 6), Oc. excrucians (n = 3), Oc. hexodontus (n = 8), Oc. intrudens (n = 14), Oc. pullatus (n = 2) and Oc. punctor/punctodes (n = 11)  regions of Finland from which collections were made, as follows: EK, Etelä-Karjala; KH, Kanta-Häme; Kl, Kymenlaakso; KP, Keski-Pohjanmaa; KS, Keski-Suomi; L, Lappi/Lapland; PK, Pohjois-Karjala; Pi, Pirkanmaa; Po, Pohjanmaa; PP, Pohjois-Pohjanmaa; U, Uusimaa; and VS, Varsinais-Suomi.Individually stored specimens were homogenised in microtubes with 100 µL of Dulbecco’s phosphate-buffered saline (PBS) + 0.2% bovine serum albumin (BSA), sterile sand and a 3 mm tungsten carbide bead . After homogenisation, the tubes were centrifuged at full speed for 5 min at 5 °C. Subsequently, 50 µL of supernatant from each specimen was then combined in a “super pool”. For pre-pooled mosquitoes, 1.8 mL of Dulbecco’s PBS + 0.2% BSA was added to each 2 mL tube, with a 5 mm tungsten carbide bead. These were homogenised using the QIAGEN TissueLyser II for 2 min at full speed, then centrifuged at 5 °C for 5 min. From each of the 91 pooled mosquito homogenates, aliquots were taken for next-generation sequencing (NGS).Prior to sequencing, the mosquito homogenates were treated following an established protocol  with minde novo assembly with the initial quality-controlled reads. Gene-like regions were detected using MetaGeneAnnotator [Sequence reads from the initial homogenates  were anannotator  and trannnotator . The aminnotator  and assiAnalyses were performed on amino acid sequences, which were derived by analysing each contig with getorf  to identThe novel viruses discovered in this study  were namEndornaviridae, Flaviviridae, Iflaviridae, Permutotetraviridae, Picornaviridae, Solemoviridae, Togaviridae and Virgaviridae. Sequences which belong to two proposed taxa, Negevirus and Quenyavirus were also recovered. The +ssRNA viruses are listed below, with all variant names and associated mosquito species in Positive-sense ssRNA viruses belonging to eight established viral families were detected during this study; Alphaendornavirus in Endornaviridae, a family of viruses known to infect plants, fungi and oomycetes, were recovered from one pool of Oc. punctor/punctodes . Complete genomes were sequenced for both virus species (GenBank accessions ON955055 and ON955056).Two species belonging to unctodes , Table 2Flaviviridae were sequenced from nine mosquito pools, four of which are tentative novel viruses . Kilpisjarvi flavivirus was most similar to Xishuangbanna aedes flavivirus  although it clustered with Ochlerotatus scapularis flavivirus  in the phylogenetic tree. The full genome of Kilpisjarvi flavivirus was sequenced (GenBank accessions ON949931). A novel flavivirus-like species, “Lestijarvi flavi-like virus”, was most similar to Hymenopteran flavi-related virus , although in the phylogenetic tree it clustered with Gudgenby flavi-like virus . Hanko virus, a species which was first described in 2012, was also present in four pools of mosquitoes collected near to the virus’ type locality, which had an average amino acid identity of >99% .Five variants of two species of  caspius , Table 2Quenyavirus, and was named “Enontekio quenyavirus”, despite being found in specimens collected from northern Lapland and from Uusimaa in the far south of Finland  and Kellev virus . Based on protein similarity, however, it clustered with Atrato Sobemo-like virus 5 . The other novel viruses, Hanko sobemovirus (amino acid identity: 83.46%), Ilomantsi sobemovirus (amino acid identity: 84.69–86.06%) and Joensuu sobemovirus (amino acid identity: 83.7–86.07%), in turn, were most similar with Atrato sobemo-like virus 4 (GenBank accession: QHA33876.1). Six sequences  was sequenced from a pool of Oc. communis collected on 26 June 2015 in Mekrijärvi, Pohjois-Karjala  were sequenced and grouped into three tentative novel species: “Hattula chuvirus” and “Kustavi chuvirus 1 and 2” ; and to Chuvirus Mos8Chu0 which was detected in Culiseta minnesotae from the USA . Kustavi chuviruses 1 and 2 were also most similar to Chuvirus Mos8Chu0 ; thus, all of the three novel species were most closely related to mosquito-derived chuviruses from the Americas.Thirteen variants from ten mosquito pools belonging to 1 and 2” , Table 3Orthophasmavirus from family Phasmaviridae were identified from six mosquito pools comprised of Oc. communis or Oc. intrudens  and Culex phasma-like virus  , mosquito-derived viruses from China and Australia, respectively. Kuusamo orthophasmavirus 1 had a low similarity to its closest matching virus, Coredo virus (amino acid identity: 41%) and Kuusamo orthophasmavirus 2 has a slightly higher similarity to Coredo virus (amino acid identity: 67.6%). Kuusamo orthophasmavirus 3 was most similar to Culex phasma-like virus  from Australia, and Kuusamo orthophasmavirus 4 to Flen bunya-like virus  from Oc. cantans that were collected in Sweden. Lastly, Lestijarvi orthophasmavirus 1 was similar to Coredo virus (amino acid identity: 64.1%) and Lestijarvi orthophasmavirus 2 to Culex phasma-like virus , the latter of which was derived from Columbian Culex.Eight novel viruses closely related to species in genus ntrudens , Table 3Phenuiviridae mainly includes arthropod-specific and vector-borne viruses that primarily infect mammals. We detected one sequence representing a novel virus belonging to genus Phasivirus and 13 phenui-like viruses (Culex mosquito [Narangue mobuvirus) . Lastly, Palkane phenui-like virus 1 also matched closely to Salari virus (amino acid identity: 69.63%), while Palkane phenui-like virus 2  shared the highest amino acid identity with Narangue virus .Family  viruses , Table 4mosquito  .Three novel variants of communis , Table 4Rhabdoviridae, viruses which infect vertebrates, invertebrates and plants, were sequenced from 13 mosquito pools and grouped into eight viruses (Merhavirus) and “Enontekio ohlsrhavirus” (Ohlsrhavirus), while the remaining species, “Enontekio rhabdovirus”, “Hattula rhabdovirus”, “Inari rhabdovirus”, “Joutseno rhabdovirus 1” and “Joutseno rhabdovirus 2” did not. Two variants of Ohlsdorf virus  were also sequenced, which were nearly identical to the originally described virus from Oc. cantans mosquitoes from Germany [Tritaeniorhynchus merhavirus) , while Enontekio ohlsrhavirus had a moderate similarity to both Ohlsdorf virus  and Riverside virus 1 (Riverside ohlsrhavirus), described from Ochlerotatus sp. mosquitoes from Hungary [Culex sp. mosquitoes in California, USA [Culex ohlsrhavirus) , Merida virus   , Ohlsdorf virus  and Perinet vesiculovirus detected in Madagascar ; Inari rhabdovirus to Ohlsdorf virus ; and both Joutseno rhabdovirus 1 and 2 to Primus virus, detected from Aedes vexans in Senegal . Complete genomes were sequenced for Enontekio merhavirus, Hattula rhabdovirus and Inari rhabdovirus .Twenty-one variants of  viruses , Table 4 Germany  , from a colony of aedine mosquitoes from Thailand; Hanko anphevirus with Serbia mononega-like virus 1  from Serbian specimens of Culex pipiens; and Joensuu anphevirus with Guadeloupe mosquito mononega-like virus  in aedine mosquitoes from Guadeloupe. The variant sequences were detected in pools of Oc. caspius, Oc. communis, Oc. hexodontus and Oc. punctor/punctodes from across Finland.hevirus” , Table 4Yueviridae is another recently validated virus family and includes viruses that have been detected from arthropods and marine diatoms. Among our specimens, we isolated one virus sequence from Oc. hexodontus, which we named “Enontekio yuevirus” (Shahe yuyuevirus) , which was sequenced from freshwater isopoda from China.uevirus” , Table 4Peribunyavirus) was identified. The sequences comprised four contigs of 301 to 630 nucleotides which mapped to the M glycoprotein segment, with >99% nucleotide identity to Russian mosquito-derived strain LEIV-15248Iv . While of a different (polymerase) gene than was included in this study, they are still of interest, as Inkoo virus is pathogenic to humans. The sequences were derived from a pool of 60 Oc. punctor/punctodes (FIN/PK-2018/11), which were collected in late June 2015.Finally, while analysing other sequence data that were generated during this study, a fragmentary genome of Inkoo virus . Eight variants were found in six pools of Oc. communis and one of Oc. intrudens .Five variants of three novel unctodes , Table 5Partitiviridae, viruses traditionally associated with fungi, plants and protozoa, but recently associated also with arthropods [Alphapartitivirus ; Ilomantsi partiti-like virus 2 with Atrato partiti-like virus 3 ; Kuusamo partiti-like virus with Culex tritaeniorhynchus partitivirus from Japan [Finally, the eight partiti-like viruses included the tentatively named “Enontekio partiti-like virus”, “Hameenlinna partiti-like virus”, “Hattula partiti-like virus”, “Ilomantsi partiti-like virus 1”, “Ilomantsi partiti-like virus 2”, “Kuusamo partiti-like virus”, “Lestijarvi partiti-like virus” and “Vaasa partiti-like virus” , Table 6Reovirales, a diverse order of viruses that infect organisms from several phyla, were sequenced , while Ilomantsi reoviruses 1–4 were moderately similar to Aedes camptorhynchus reo-like virus . However, a phylogenetic analysis suggested that Atrato reo-like virus (GenBank accession: QHA33824.1) was more related to Ilomantsi reoviruses 1–3, while Ilomantsi reovirus 4 clustered near the root of the Ilomantsi reovirus clade.Five novel reoviruses belonging to equenced , Table 7Totiviridae, which includes viruses of fungi and protozoans, among others. From 205 sequences, 52 viruses were identified, of which 50 were novel and two were strains of previously described, albeit unnamed, viruses . Similarly, Enontekio totivirus 2, Hanko totiviruses 8 and 9, Hattula totivirus 2, Ilomantsi totivirus 3, Joutseno totivirus and Vaasa totivirus had a low to moderate amino acid identity to an unnamed dsRNA from an environmental sample . Hameenlinna totivirus 3 sequences had a low amino acid identity with multiple previously established viruses including the aforementioned unnamed virus (amino acid identity: 36.36–54.53%), Aedes aegypti toti-like virus , Emileo virus  and Hubei toti-like virus 10 . Enontekio totivirus 3 was also similar to Hubei toti-like virus 10 (amino acid identity: 56.29%). The most viral sequences in this study grouped within  viruses , Table 7Enontekio totiviruses 4 and 5 as well as Hattula totivirus 3 were most similar to Murri virus . Hattula totivirus 3 also shared amino acid identity with Atrato virus , although based on our phylogenetic tree, the virus in general was more related to Murri virus. Enontekio totiviruses 6 and 7 had the most similarity with Beihai razor shell virus 4 . Thirteen viruses shared a moderate amino acid identity with an unnamed uncultured virus (GenBank accession: AGW51771.1). These were Hameenlinna totiviruses 1 and 2 (amino acid identities: 48.73–65.96% and 62.42–70.73%), Hanko totiviruses 5, 7 and 10 , Ilomantsi totiviruses 1 and 2 (amino acid identities: 54.61–64.58% and 59.68–72.16%), Inari totivirus 1 (amino acid identity: 48.98–57.46%), Karstula totivirus (amino acid identity: 41.18%), Kuusamo totiviruses 1 and 2 (amino acid identities: 73.75% and 45.61%), Lestijarvi totivirus (amino acid identity: 39.84–42.51%) and Palkane totivirus (amino acid identity: 60.16–61.96%). Despite sharing the highest amino acid identity with the uncultured virus, Karstula totivirus, Hanko totivirus 10 and Kuusamo totivirus 2 clustered with Fushun totivirus 4 and Sanya totivirus 7 . Hattula totivirus 1 was similar to Pisingos virus , Hanko totivirus 2 to Erysiphe necator associated totivirus 7  and Hanko totivirus 6 to Aedes alboannulatus toti-like virus 1 . Other detected totiviruses that shared similar amino acid identities to Aedes alboannulatus toti-like virus 1 were strains of the aforementioned Hameenlinna totivirus 1 (amino acid identity: 62.6%) and Inari totivirus 1 (amino acid identity: 62.2%). Several strains of our novel totiviruses were moderately similar to Aedes camptorhynchus toti-like virus 1 (GenBank accession: YP_009388611.1), Inari totivirus 2 (amino acid identity: 52.88–53.19%), Hameenlinna totivirus 1 (amino acid identity: 60.98%) and Lestijarvi totivirus (amino acid identity: 53.3%). Lastly, Hanko totivirus 1 shared a low amino acid identity with Malassezia sympodialis mycovirus .Two totivirus sequences were Finnish strains of a previously described “uncultured virus” from France (GenBank accession: AGW51771.1), which have nearly identical protein identities. In the absence of a name, these viruses were therefore tentatively named “Hanko totivirus 3” and “Hanko totivirus 4” (amino acid identities: 96.76–97.35% and 94–94.5%). Complete genomes were detected for 16 aforementioned totiviruses, as follows: Enontekio totiviruses 2 and 5, Hameenlinna totiviruses 1 and 3, Hanko totiviruses 3, 5, 8 and 9, Hattula totiviruses 1 to 3, Ilomantsi totivirus 1, Inari totiviruses 1 and 2, Joutseno totivirus and Lestijarvi totivirus.Seventeen novel toti-like viruses were recovered, which did not cluster within any established genera. As such, they were provisionally named “Enontekio toti-like virus 1 to 4”, “Hameenlinna toti-like virus”, “Hanko toti-like virus 1 to 3”, “Ilomantsi toti-like virus 1 to 3”, “Inari toti-like virus”, “Kustavi toti-like virus”, “Kuusamo toti-like virus”, “Palkane toti-like virus”, “Utsjoki toti-like virus” and “Vaasa toti-like virus”. Based on amino acid identity, Enontekio toti-like virus 1 was distantly similar to Uromyces totivirus D , yet clustered with Uromyces potyvirus A (GenBank accession: QED42911.1). Enontekio toti-like viruses 2 and 3 had a low similarity to diatom-colony-associated dsRNA virus 7 , while Enontekio toti-like virus 4 to diatom-colony-associated dsRNA virus 11 . Seven of the viruses matched Nuyav virus (GenBank accession: QRW41699.1). These included Hameenlinna toti-like virus (amino acid identity: 68.09–73.6%), Hanko toti-like virus 2 (amino acid identity: 68.98%), Ilomantsi toti-like viruses 1 and 2 (amino acid identities: 76.01% and 66.82–71.27%), Kustavi toti-like virus (amino acid identity: 88.62–91.38), Palkane toti-like virus (amino acid identity: 72.59–76.81%) and Vaasa toti-like virus (amino acid identity: 68.53–69.73%). Ilomantsi toti-like virus 3 were distantly similar to an unnamed RNA virus . Hanko toti-like virus 1 was distantly related to Keenan toti-like virus  and according to the phylogenetic analysis  to two DVariable numbers of pools, ranging from 1 to 35, were prepared for each mosquito species included in this study, with pooled material obtained from multiple collection locations. This made direct comparison of some results between species less meaningful, but each species was associated with multiple viruses.Ochlerotatus cantans, which was the least represented species in the study with only one pool of 20 specimens collected in late June 2015 in Ilomantsi, PK, was found to have six viral sequences. These represented five novel species, and clustered within Solemoviridae, Partitiviridae and Totiviridae  and Sindbis virus . This is the first confirmed mosquito species to be associated with Sindbis virus in Finland. The single strain was found in Mekrijärvi, Pohjois-Karjala, an area where the only other Finnish mosquito-borne Sindbis virus strains have been recovered. The remaining 58 novel species belong to Aliusviridae, Botybirnavirus, Chrysoviridae, Chuviridae, Iflaviridae, Negevirus, Partitiviridae, Phasmaviridae, Phenuiviridae, Qinviridae, Sedoreoviridae, Rhabdoviridae, Solemoviridae, Totiviridae, Virgaviridae and Xinmoviridae  pools, which were collected from northern and western Finland in June and July 2015. Twenty sequences were assembled, which grouped into 12 virus species, 11 of which were novel. Two strains of the previously described Ohlsdorf virus (Rhabdoviridae) were found from Inari in Lapland. The other 11 species grouped within Negevirus, Partitiviridae, Permutotetraviridae, Solemoviridae and Totiviridae  and Cordoba virus (Negevirus) (gevirus) . Short MOchlerotatus mosquitoes, thereby ascertaining both the diversity of associated viruses and the potential vector associations of these mosquito species. RNA sequences were generated from nine identified species of female Ochlerotatus (2333 specimens), which were divided into 91 species-specific pools. Viral sequences were present in all mosquito pools, but only 90 contained sequences of RdRp greater than 1000 nucleotides and were included in further analyses. In total, 514 viral RNA sequences were identified that grouped into 159 species, 147 of which were likely to be novel. Strains for 12 viruses which had previously been described were sequenced, although only nine had been named when published: Hallsjon virus (Endornaviridae), Hanko virus (Flaviviridae), Cordoba virus, Dezidougou virus and Mekrijärvi negevirus (Negevirus), Jotan virus (Picornaviridae), Ohlsdorf virus , Evros sobemo-like virus (Solemoviridae) and Sindbis virus (Togaviridae). The remaining three unnamed viruses were given suggested names in this study, to correspond with where the Finnish sequences originated: Hanko picorna-like virus (Picornaviridae), Hanko totivirus 3 and Hanko totivirus 4 (Totiviridae). Only two of these previously described viruses are currently recognised by the ICTV: Sindbis virus and Ohlsdorf virus. Three viruses, which had previously been detected using virus cell culture with Finnish mosquitoes, were sequenced and linked to named mosquitoes as follows: Hanko virus with Oc. caspius, Inkoo virus with Oc. punctor/punctodes and Sindbis virus with Oc. communis. These results affirm the high degree of viral diversity found in mosquitoes from Finland, despite only nine of the forty-three endemic mosquito species [This is the first in-depth study of the viromes of mosquitoes from Finland. The aim was to investigate RNA viromes of identified  species  being inConstructing phylogenetic trees of RNA viruses using RNA-dependent RNA polymerase sequences is a common practice to infer evolutionary relationships and classify newly detected viruses. This is because RdRp is a core viral protein which has conserved sequence motifs that make it a preferable gene to utilise in phylogenetic analyses. The nucleotide sequences of RNA viruses change constantly due to the high mutation rate in RNA viruses, but in contrast, amino acid sequences remain relatively stable and conserved . PhylogeThe putative viruses sequenced in this study were assigned as novel based on several criteria: (1) novel virus RNA dependent RNA polymerases submitted to NCBI BLASTx had to have an amino acid identity value lower than 90% compared to the most similar virus; (2) phylogenetic analyses were run to ascertain their evolutionary relationship with previously described viruses and their likely classification within virus families; (3) associated GenBank records from closely related taxa were examined and compared with potentially novel viruses. These included the country of sampling, collection date and the organism from which the virus was isolated, to infer their novelty. Finally, (4) the criteria set by ICTV, including sequence lengths, amino acid identity and clustering in phylogenetic trees were also considered. Additionally, we computed supplementary pairwise distances from the protein alignments to ascertain the novelty of the detected viruses . CertainSpinareoviridae), but low values were encountered many times throughout the analysis. This highlights the issues presented by “viral dark matter”, i.e., the lack of available sequences in databases to which viral sequences can be aligned [Most (147) of the 159 viruses reported in this study were designated as novel since they had low similarities in RdRp amino acid identity with the most similar existing viruses (average 65.88%). The lowest amino acid identity was seen with Enontekio reovirus, which was only 29.6% similar to Operophtera brumata reovirus  were affected by these strict parameters, since short contigs containing polymerase, glycoprotein and nucleocapsid sequences were recovered.Many more new viruses could have been named from the sequences obtained from this study but were excluded as their contigs fell below the 1000-nucleotide minimum length requirement that was set for any sequences to be considered for analysis. These discarded sequences formed approximately 75.5% of the total viral sequence data generated. In particular, short sequences of the pathogenic species Inkoo virus and Aedini, a cosmopolitan tribe of Culicidae with 1263 extant species and which includes 35% of all valid mosquito species [Flaviviridae (Flavivirus) clustered near to or with insect-specific and Aedini-associated viruses. Several of the novel viruses which grouped within Picornaviridae, Chuviridae and Chrysoviridae were the first mosquito-associated viruses detected which belonged to tribe Aedini. These findings could be indicative of broader mosquito association ranges among these RNA virus families. Among the virus families which infect plants and fungi, e.g., Alphapartitivirus, the discovery of these novel Finnish viruses would suggest that Ochlerotatus mosquitoes  act in some capacity as vectors for these viruses, whether by mechanical transmission or otherwise.A common pattern observed in the phylogenetic trees was that novel viruses clustered with available sequences of mosquito-derived viruses, inferring that these might be more mosquito-specific than insect-specific. Moreover, many novel viruses obtained during this study clustered with viruses that were sequenced from other mosquitoes, many of which belonged to  species . One exp species . Viral sOchlerotatus pools was very high in this study ). GenBank records show that totiviruses have been found in arthropods, plants, mammals and fish, thus indicate that these viruses might have a wider host range than is currently recognised by the ICTV ). Another factor to potentially explain the high prevalence of totiviruses could be that they are part of the core virome of Ochlerotatus species [Totiviridae to a critical review, since at present only 28 species belonging to five genera are currently officially recognised by the ICTV ), but in this study alone, 52 novel viruses were proposed. Similarly, partitiviruses were the next most represented species in this study, with 52 strains belonging to 23 viruses.The proportion of totivirus sequences detected in all is study , despite species . Either In recent years, most of the novel mosquito-borne viruses have been detected and reported from temperate and equatorial regions, since that is where most of the known mosquito-borne diseases are distributed . The numChrysoviridae, Endornaviridae, Solemoviridae, Totiviridae and Virgaviridae are more traditionally associated with protozoa, plants or fungi than mosquitoes  and two Oc. punctor/punctodes (FIN/PP-2018/015 and FIN/L-2018/026) were noted to have parasitic or phoretic mites attached to them . If truly phoretic, then the mites may just have been temporarily attached to the mosquito for dispersal, so they may not have been so relevant for interspecies transmission. If, however, they were parasitic, then the transfer of viruses between mites and mosquitoes is not out of the realm of possibility [Mosquitoes also have many interactions with other organisms in the environment. Some species are known to feed on honeydew, a sugar-rich excrement that some insects including ants (Hymenoptera) and aphids (Hemiptera) excrete after feeding on plants ,93. It wsibility . More wo® Elute Cards , have been used in several studies in recent years in order to collect mosquito saliva, preserve any viral RNA, and ultimately sequenced to determine which viruses/virus species are present [Taking these points into consideration, a further laboratory step would have also increased our understanding of which viruses may be vectored by the mosquitoes included in these analyses. Honey-baited nucleic acid cards, such as FTA present ,96,97. BOc. cantans in Germany, Ohlsdorf virus [Rhabdoviridae), and six novel viruses belonging to Partitiviridae (1), Permutotetraviridae (1), Solemoviridae (1) and Totiviridae (3). Pool FIN/L-2018/27 only contained a single virus, Hanko iflavirus 1 (Iflaviridae). Pool FIN/L-2018/88 contained eight novel viruses, which belonged to Chrysoviridae (1), Partitiviridae (1), Sedoreoviridae (1), Totiviridae (4) and Virgaviridae (1). Competition between different viruses within mosquitoes might inhibit the replication or transmission of other viruses, resulting in the over representation of more competitive viruses [When mosquitoes were collected for this study, a note was made whenever a female was noticeably blood fed or gravid, but not if they had distended abdomens which looked as though they had recently fed upon plant juices. All but three of the females were not visibly blood-fed with only one female from pools FIN/L-2018/07 and FIN/L-2018/27 being confirmed as such, and one female which looked like it had possibly blood-fed several days earlier, in pool FIN/L-2018/88. Pool FIN/L-2018/07 contained seven viruses, one described from rf virus   albopictus [Culex mosquitoes [Ae. albopictus [Ae. albopictus mosquitoes, in which Aedes iflavi-like virus genomes were only detected in a pool of male mosquitoes [Viromes of other mosquitoes which are native to Finland would also be of interest to study further in the future. This study only included nine of 43 (21%) currently recognised endemic species , and 38%bopictus  and Culesquitoes , though bopictus . The solsquitoes . The autsquitoes .Sindbis virus from Pohjois-Karjala [This study has significantly increased the number of locations from which virus-positive mosquitoes have been collected in Finland. Prior studies have detected Hanko and Inkoo viruses from Uusimaa ,4,8, Lam-Karjala ,2 and fi-Karjala . In all,-Karjala ,9.In contrast, this study included specimens which were collected from 49 collection efforts at 43 sites (min 1 km separation) in 11 regions and extended the sampling locations of virus-positive mosquitoes to the entire country see . MoreoveOc. caspius, which were collected in late August 2017. This is the first instance where a named mosquito species is confirmed to be associated with the virus. With future analyses, it will be interesting to see if Hanko virus is restricted to Oc. caspius, a halophilic/coastal mosquito species [Oc. caspius were included in the analysis, from Kustavi in Varsinais-Suomi from collections made in July and August 2017  and Varsinais-Suomi (Southwest Finland). This, however, is very likely explained by sampling bias, since only one mosquito pool included specimens from Keski-Suomi and four pools from Varsinais-Suomi.A disproportionate number of pools were comprised of specimens which were collected from around Mekrijärvi, or in the municipality of Ilomantsi, Pohjois-Karjala. This was in part because the material in this study was all snap-frozen, identified and stored at −70 °C immediately following identification, to permit virus cell culture experiments. Such specialist facilities are located at a few field stations around Finland, which also explains why many collections were also made around the municipalities of Enontekiö and Utskoki in Lapland and in Hanko, Uusimaa. There were other factors, however, which influenced the decision to include material from eastern Finland. Prior to this study, Pohjois-Karjala was the only region where is virus ,2, and ois virus , and vecpulation , but virCoquillettidia richiardii, Oc. communis, Oc. annulipes, Oc. cantans, Culex pipiens and Cx. torrentium, all species which are common to both Sweden and Finland, and two of which were common to both studies. They found viruses which belonged to multiple families, but ultimately there were none that were common to both studies [Nodaviridae, Orthomyxoviridae, Tombusviridae and Articulavirales [Culex pipiens and Cx. torrentium collected from two locations over several years. They found 40 viruses (28 novel viruses) belonging to 14 families/orders: Bunyavirales, Endornaviridae, Luteoviridae, Mogonegavirales, Negevirus, Nidovirales, Orthomyxoviridae, Partitiviridae, Picornaviridae, Qinviridae, Reoviridae, Togaviridae  Totiviridae and Virgaviridae [Sindbis virus, Hallsjon virus and Jotan virus were common to both this and the Swedish study [Luteoviridae and Orthomyxoviridae were not sequenced in this study.Metagenomics studies published in recent years have identified diverse viromes in mosquitoes from around the world ,111,112. studies . They diavirales . Anotheraviridae . Sindbissh study , but virFlaviviridae, Iflaviviridae, Tymoviridae, Bunyavirales, Rhabdoviridae, Partitiviridae, Totiviridae and Metaviridae. Lampyris noctiluca flavivirus 1 grouped within the same clade as Lestijarvi flavi-like virus in the Flavivirus tree  amplified targeted RNA sequences from adults collected in central and southern Finland . They rerus tree , in a brrus tree , in a brPeribunyaviridae: Orthobunyavirus) and Sindbis virus (Togaviridae: Alphavirus) have known disease associations in Finland, and, although infrequent, can cause severe enough symptoms for patients to require hospitalisation [Oc. punctor/punctodes and Oc. communis, respectively. The first isolations of Inkoo virus in the 1960s did include mixed pools containing Oc. communis and Oc. punctor/punctodes but now Oc. punctor/punctodes is confirmed as being virus positive. While most of the detected viral diversity has not been, and likely will not be, associated with pathogenic traits, it is nevertheless notable that without targeted sampling to capture outbreaks spatially or temporally, we have been able to detect sequences of the two previously well-established mosquito-borne pathogenic viruses in Finland.Two of the viruses which were sequenced in this study, Inkoo virus  is an order comprised of two families, Sedoreoviridae and Spinareoviridae (formerly Sedoreovirinae and Spinareovirinae), each of which has pathogenic virus species among their members. It is for this reason that the five novel Reovirales viruses in this study are of particular interest for future examination, to determine if hitherto unrecognised pathogenic mosquito-borne viruses are present in Finland. The proposed Sedoreoviridae viruses (Ilomantsi reovirus 1 to 4) all group with other viruses which were sequenced from mosquitoes, and are related to Phytoreovirus, which includes plant-pathogenic viruses based on a phylogenetic analysis. Valmbacken virus (see Spinareoviridae virus, Enontekio reovirus, is distantly related to Fijivirus, which includes plant-infecting viruses that may spread via an insect vector. The sequences which group together in irus see , which iThis study, by using high throughput next-generation sequencing methods and an unbiased virus discovery pipeline, has vastly increased the knowledge of viruses associated with mosquitoes in Northern Europe and has confirmed the number of known mosquito-associated viruses and virus families in Finland from seven and four, to 159 and 25, respectively. Such a large increase in knowledge of the diversity of mosquito-associated viruses is certainly interesting and begins to enlighten the “viral dark matter”, but inevitably brings with it new questions and challenges. It also highlights the pressing need for additional study to bring relevance to the names and sequences presented herein, as well as to investigate arthropod viromes of northern regions more thoroughly. It is evident from the points we have raised that the floodgates have opened, and the real work of elucidating the relationships between mosquitoes, viruses, the environment and host species must now begin.


Data on the impact of the cumulative percutaneous left atrial appendage closure (LAAC) caseload on cardiovascular outpatient and hospitalisation costs are limited.The present single-institution analysis includes patients treated consecutively from the beginning of our LAAC experience in January 2012 until December 2016. Pre- and post-LAAC costs for hospitalisation and ambulatory visits were included.p = 0.8) and there was no correlation between cumulative caseload and procedural costs (p = 0.9). Although annualised cardiovascular management costs after LAAC were lower than before LAAC  from the beginning of the cumulative procedural experience, a significant reduction in costs was observed only from 2014 onwards. Institutional cumulative LAAC caseload and year of procedure were not related to the amount of reduction in the costs for cardiovascular care.A total of 676 patients underwent percutaneous LAAC (using the Watchman device): 49 (2012), 78 (2013), 211 (2014), 210 (2015), and 129 (2016). LAAC procedural costs were stable over the years (overall median €9639; 2012: €9630; 2013: €10,003; 2014: €9841; 2015: €9394; 2016: €9530; LAAC led to cost-of-care savings from the beginning of our institutional procedural experience. We are the first group to investigate actual real-world management costs of percutaneous left atrial appendage closure (LAAC) in patients with non-valvular atrial fibrillation and to focus on the impact of the learning curve.Management costs are not learning curve related.Cost savings  have been significant and evident since our very early clinical experience with the procedure.Moreover, we are the first group to show an independent relation (inverse) between DRG clinical complexity level and cost-of-care savings after the procedure.Finally, we have shown that survival after LAAC is not affected by the caseload or procedural experience.Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia with a risk of cerebral thromboembolism that rises with the increase in patients’ age and cardiovascular comorbidity , 2.Because the left atrial appendage (LAA) is the most common and clinically relevant source of thrombi in patients with non-valvular AF, its percutaneous closure (LAAC) represents a valid armamentarium in patients with an absolute contraindication to anticoagulation.Percutaneous LAAC is still not widely implemented in daily clinical practice and is considered a novel procedure. Therefore, outcomes and costs could be impacted by the cumulative LAAC caseload and by the learning curve. In the current analysis we have investigated the impact of the cumulative LAAC caseload on costs, cost-of-care savings, and outcomes. In particular we report on the pre-, intra-, and post-procedural management costs in a consecutive series of patients with thromboembolic non-valvular AF treated with percutaneous LAAC using the Watchman device .The Vivantes network of affiliated hospitals in Berlin is the largest community-based hospital chain in Germany, serving an area that includes about one-third of the circa 3.6 million inhabitants of Berlin.Percutaneous LAAC has been performed within the Vivantes network since January 2012, using the Watchman device. The present analysis includes only consecutive patients treated with LAAC between January 2012 and December 2016. Hospital admissions (including ambulatory visits) to the Vivantes clinics occurring before and after the admission for percutaneous LAAC were identified. Only hospital admissions and ambulatory visits for cardiovascular reasons, including anticoagulation and bleeding management, were selected, and costs were retrieved. The observation period spanned from the first admission with a diagnosis of AF until the last admission, for follow-up post-LAAC or management of cardiovascular complications, or other issues related to LAAC procedure/management.Demographic, clinical, and treatment cost information regarding the admitted patients was prospectively recorded in a centralised electronic database and was retrospectively analysed.2DS2-VASc scores ) were computed.HAS-BLED  and alcohol (more than eight drinks a week)) and CHAHospital service fees were calculated and remunerated via the DRG (diagnosis-related groups) system. Details of the DRG’s remuneration system are regulated by the Hospital Financing Act, the Hospital Remuneration Act, and the flat rate case agreement of the self-government partners.For every patient, the primary diagnosis and secondary diagnoses were recorded and used to compute cumulative and yearly costs for hospital admissions and visits before LAAC, the LAAC procedure itself, and after LAAC. The clinical complexity level  was used, together with the primary diagnosis, to account for the severity of the comorbidities and complications in the secondary diagnoses and for billing to the DRG system. The CCL weighting is divided into four levels according to the comorbidity and morbidity profile .The ethics committee of the University of Applied Sciences of Neubrandenburg  approved the study under the registration number HSNB/KHM/146/19.Patients were classified according to the year of the LAAC procedure year  and a consecutive case number was also assigned starting from the first procedure (continuous variable).Overall costs, procedural costs, pre- and post-LAAC total and yearly  costs were calculated. Differences between pre- and post-LAAC costs were also computed to identify the possible cost-of-care savings after LAAC.Analysis of variance (ANOVA) (post hoc Tukey test), non-parametric tests, chi-square and Fisher exact tests were used when appropriate and to compare differences among year of LAAC procedure groups. Multivariate analysis by means of backward linear regression were used to identify independent determinants of cost-of-care savings after LAAC.Kaplan-Meier survival curves were generated to calculate estimated survival at follow-up and differences in survival according to the year of the LAAC procedure (log rank test).p < 0.05 (two-tailed).Data are presented as absolute numbers, percentages, mean ± standard deviation, or median with 75% interquartile range (IQR) as appropriate. Statistical analysis was performed using IBM SPSS version 25  and the level of significance was set at p < 0.0001). Fig. p = 0.5). A significant reduction of management costs was observed only from 2014 onwards , 78 (2013), 211 (2014), 210 (2015), and 129 (2016). Tab. 2, 78 (20p = 0.4).The median follow-up duration after LAAC was 4.8 years (IQR: 3.6–5.6). Observed all-cause follow-up mortality was 16.9%. Kaplan-Meier analysis revealed no differences in survival between groups stratified by year of implantation Fig. ; p = 0.4Linear regression analysis revealed that only CCL was an independent determinant of the amount of cost-of-care savings after LAAC (inverse relationship) Fig. .Fig. 3LiCosts are not increased in the initial phase of the learning curve with LAAC. On the contrary, cost-of-care savings are evident from the very early phase of our clinical experience with percutaneous LAAC.There is an inverse and independent relation between CCL, computed using the DRG indications, and cost-of-care savings after LAAC.Survival after LAAC is not affected by caseload/procedural experience.From our actual and real costs analysis, which considers the development of our institutional experience, it emerges that:To the best of our knowledge, we are the first group to investigate actual real-world management costs of percutaneous LAAC and focus on the potential impact of the procedural learning curve. In the current scientific literature, economic evaluations of percutaneous LAAC are derived mainly from mathematical simulations and very seldom from documented costs originating from analysis of DRG documents for specific timeframes –5. In thThe analysis presented herein is the first to demonstrate that outpatient and clinical care costs related to non-valvular AF are not affected by the institutional learning curve with percutaneous LAAC. The reported costs are actual amounts claimed from the German health insurance funds. These costs were derived from the DRG system and hospital claims records and are not based on mathematical modelling or any other assumptive calculations.Since the introduction of percutaneous LAAC in our hospital network in 2012, procedural costs have been stable, and medical management costs have significantly decreased after treatment, justifying the procedure costs in selected patients with a contraindication to oral anticoagulation. We have previously confirmed that patients undergoing LAAC will cause lower AF-related hospital costs secondary to admissions and outpatient treatments to manage anticoagulation, its iatrogenic effects, and to diagnose and treat cerebrovascular embolism . In factAs documented in our study, cost-of-care savings are related to the clinical complexity of the single patient, represented in the DRG CCL value, rather than to the institutional cumulative caseload.The volume-outcome relationship has been proposed for complex surgical procedures and percutaneous treatment of structural heart disease. In particular, a difference in mortality and hospitalisation costs, based on the annual procedural volume, has emerged within groups of hospitals performing transcatheter aortic valve replacement . In the Finally, we are aware that in the existing literature, management costs for LAAC patients have been compared with costs in patients managed solely with oral anticoagulation. We believe that such a comparison risks including patients with very different risk profiles and conceals the impact of the treatment strategy. In light of this, we have preferred to use every patient as her/his own control and to evaluate the variation in management costs after percutaneous LAAC. Such an analysis was possible only because our network of affiliated clinics serves the large majority of the urban population of Berlin, and, for this reason, we have a very granular picture of treatments performed per single patient over the years.In the present work we have shown that since the beginning of our experience with LAAC in 2012, the LAAC implantation costs have remained stable and there has been a constant post-procedural cost-of-care saving. Management costs and cost-of-care savings are independent of the institutional cumulative caseload. These findings are supported by the fact that peri-procedural complications and mortality have remained limited, even at the beginning of our learning curve, and follow-up outcomes have confirmed the safety of this innovative technology.


Numerical stabilization is often used to eliminate  the spurious oscillations generally produced by full order models (FOMs) in under‐resolved or marginally‐resolved simulations of convection‐dominated flows. In this article, we investigate the role of numerical stabilization in reduced order models (ROMs) of marginally‐resolved, convection‐dominated incompressible flows. Specifically, we investigate the FOM–ROM consistency, that is, whether the numerical stabilization is beneficial both at the FOM and the ROM level. As a numerical stabilization strategy, we focus on the evolve‐filter‐relax (EFR) regularization algorithm, which centers around spatial filtering. To investigate the FOM‐ROM consistency, we consider two ROM strategies: (i) the EFR‐noEFR, in which the EFR stabilization is used at the FOM level, but not at the ROM level; and (ii) the EFR‐EFR, in which the EFR stabilization is used both at the FOM and at the ROM level. We compare the EFR‐noEFR with the EFR‐EFR in the numerical simulation of a 2D incompressible flow past a circular cylinder in the convection‐dominated, marginally‐resolved regime. We also perform model reduction with respect to both time and Reynolds number. Our numerical investigation shows that the EFR‐EFR is more accurate than the EFR‐noEFR, which suggests that FOM‐ROM consistency is beneficial in convection‐dominated, marginally‐resolved flows. However, FOMs are generally not accurate in the under‐resolved regime, that is, when the number of degrees of freedom is too low to represent the flow dynamics. We note that FOMs are also inaccurate in the marginally‐resolved regime, when the number of degrees of freedom is just enough to capture the flow dynamics. However, the FOM inaccuracy is less pronounced in the marginally‐resolved regime than in the under‐resolved regime. A classic illustration of under‐resolved and marginally‐resolved FOMs is in the convection‐dominated regime , when the FOM mesh‐size is larger than the smallest flow scales. In this case, the FOMs generally yield spurious numerical oscillations that significantly degrade the FOM numerical accuracy.In the numerical simulation of incompressible fluid flows modeled by the Navier–Stokes equations (NSE), standard full order models (FOMs)  methods, finite volume (FV) methods, finite difference (FD) methods or spectral elements (SE)) work well in the numerical stabilization methods have been proposed over the years. Many of these FOM numerical stabilization approaches are surveyed in the research monograph of Roos et al..Regularized models are a popular class of numerical stabilization methods in which spatial filtering is used to regularize (smooth) various terms in the NSE and eliminate  the spurious numerical oscillations of the FOMs in convection‐dominated, under‐resolved and marginally‐resolved simulations. A plethora of regularized models are surveyed in the research monograph of Layton and Rebholz.Leray model (proposed in 1934 by the great mathematician Jean Lerayevolve‐filter‐relax (EFR) model, which consists of three steps: (i) in the “evolve” step, a standard FOM is used to obtain an intermediate approximation of the velocity; (ii) in the “filter” step, a spatial filter is used to filter (regularize) the intermediate approximation obtained in step (i) and eliminate  its spurious numerical oscillations;simplicity and modularity: given a legacy FOM code, the “evolve” step is already implemented, the “filter” step requires the addition of a simple subroutine, and the “relax” step is just one line of code.To eliminate or alleviate the spurious numerical oscillations of the FOMs in convection‐dominated, under‐resolved and marginally‐resolved simulations, various To summarize the above discussion, when FOMs are used in the convection‐dominated, under‐resolved  regime, regularized models  can be used to eliminate  the spurious numerical oscillations. In general, the need for numerical stabilization in the convection‐dominated, under‐resolved  regime is well known and well documented in the realm of classical numerical methods: there are hundreds (if not thousands) of papers and several research monographs on this topic, and commercial software often includes numerical stabilization strategies for the under‐resolved  regime. Our goal is to investigate this topic in a reduced order modeling context.Reduced order models (ROMs) are relatively low‐dimensional computational models that can reduce the FOM computational cost by orders of magnitude.(Q1)Assuming that the FOM is run in the convection‐dominated, resolved regime and the ROM is run in the convection‐dominated, under‐resolved  regime, is numerical stabilization needed for the ROM?regularized ROMs (Reg‐ROMs), for example, the Leray ROMFor Galerkin projection based ROMs, it was shown in References A natural follow‐up question to (Q1) is the following:(Q2)Assuming that both the FOM and the ROM are run in convection‐dominated, marginally‐resolved regime, if numerical stabilization is used for the FOM, is numerical stabilization still needed for the ROM?(i)EFR‐noEFR, in which we use the EFR regularization at the FOM level but not at the ROM level; and(ii)“FOM‐ROM consistency”.EFR‐EFR, in which we use the EFR regularization both at the FOM level and at the ROM level. In this article, we call this strategy To our knowledge, question (Q2) is still open. In this article, we take a step in answering question (Q2). Specifically, we consider two scenarios in which both the FOM and the ROM are run in the marginally‐resolved regime. In this setting, we compare two types of ROMs:We compare the EFR‐noEFR and the EFR‐EFR in the numerical simulation of a 2D incompressible flow past a circular cylinder with time‐dependent Reynolds number.The main difference between the two investigations is the algorithm used: we use the EFR algorithm, whereas in Reference An important difference between the two investigations is that in the current study we perform the model reduction both in time and in the parametric space. Specifically, we leverage a nested proper orthogonal decomposition (POD) approach to develop ROMs that include variations with respect to the Reynolds number, which is a critical parameter in practical ROM applications. In contrast, the investigation in Reference Another significant difference between the two investigations is the spatial discretization at a FOM level: in the current study we employ the FE method, whereas the investigation in Reference To our knowledge, this is the first time the FOM‐ROM consistency is investigated for the EFR regularization. The FOM‐ROM consistency has been advocated only in a few other settings, for example, for classical residual based stabilization methodsRemark 1Although enforcing FOM‐ROM consistency may seem a natural choice,The reason for the relative popularity of the FOM–ROM inconsistency is most probably its practical convenience: to build the ROM, one is not restricted by the particular choices made in the FOM numerical discretization. Thus, the FOM–ROM inconsistency belongs to the general class of approaches that consider the FOM data and the ROM as different entities. In this article, we espouse a different line of thought in which the FOM and ROM are not completely independent. Specifically, we show numerically that building ROMs that are consistent with the FOMs with respect to the particular numerical regularization used can yield more accurate solutions.The rest of the article is outlined as follows: In Section 2Ω⊂ℝD, with D=2,3, we consider the motion of an incompressible fluid having velocity u≐u∈𝕌 and pressure p≐p∈ℚ represented by the NSE:u=u0 in Ω×{t0}, where ∂ΩD∪∂ΩN=∂Ω, ∂ΩD∩∂ΩN=∅, ν is the kinematic viscosity, and 𝕌 and ℚ are suitable Hilbert function spaces. The functions uD and u0 are given.In this section, we present the FOM and the EFR algorithm. As a mathematical model, we use the incompressible Navier–Stokes equations (NSE). Given a fixed domain U and L represent the characteristic velocity and length scales of the system, respectively. When the Reynolds number is large, the inertial forces dominate the viscous forces; this setting is generally referred to as the convection‐dominated regime. As explained in the introduction, it is well known that in the convection‐dominated regime standard spatial discretizations yield spurious numerical oscillations in under‐resolved and marginally‐resolved numerical simulations. In our numerical investigations in Sections The flow regime is defined by the Reynolds numberu∈𝕌Nhu and p∈ℚNhp, respectively, where Nhu and Nhp are the corresponding numbers of degrees of freedom. We denote the time step with Δt. Let tn=t0+nΔt for n=0,…,NT, and T=t0+NTΔt. We denote with yn the approximation of a generic quantity y at the time tn. The EFR algorithm at the time tn+1 yields: χ∈ is a relaxation parameter. Here w is the evolved velocity and w‾ is the filtered velocity. We note that using wn+1=un+1 in step (I)  is equivalent to solving the NSE. In step (II), we use a differential filter (DF) with an explicit length scale, δ, which is the filtering radius . The success of DFs is due to several appealing properties.χ diminishes the magnitude of the numerical diffusionχ∼Δt is commonly used.In what follows, we denote the semidiscrete FE velocity and pressure with 3In this section, we focus on our POD‐Galerkin ROM framework for model reduction with respect to time. In Section 3.1{φj}j=1r and {ψj}j=1r, for the reduced velocity and pressure spaces 𝕌r and ℚr, respectively, so that{aju(t)}j=1r and {ajp(t)}j=1r are the sought time‐varying coefficients.{ui}i=1Nu⊆{uk}k=1NT and {pi}i=1Np⊆{pk}k=1NT, where Nu and Np denote the number of snapshots for velocity and pressure, respectively. We utilize the EFR at the FOM level to generate the snapshots for both the EFR‐noEFR and the EFR‐EFR strategies. We employ the POD algorithmThe basic idea of ROMs is to build a low‐dimensional framework where the problem at hand can be solved more efficiently than the FOM. To this end, assume that we have two bases, supremizer stabilization for the reduced velocity space in order to guarantee the well‐posedness of the system. We emphasize that the main role of the supremizers is to avoid spurious reduced pressure modes. To tackle the convection‐dominated, marginally‐resolved regime, different approaches are needed. The supremizer stabilization proposed in Reference S:ℚNhp→𝕌Nhu defined as{pi}i=1Nu in i=1Nu. However, a standard POD procedure is applied to {pi}i=1Np: rp POD eigenpairs are retained to build the bases. The supremizer technique leads to a reduced velocity space of dimension rus=ru+rs. We denote the enriched reduced velocity dimension with rus, and consider {φj}j=1rus as the enlarged velocity‐supremizer basis. We define the pressure basis as {ψk}k=1rp.It is well known that, in a standard NSE setting, the POD may be combined with a pace is𝕌rus≐POD3.2tn+1, find the pair  that solvesi=1,…,rus, and j=1,…,rp. Algebraically, we are looking for the (n+1)‐st solution ofun+1∈ℝrus and pn+1∈ℝrp are the vectors of the reduced coefficients of r is negligible and thus the costs of the EFR‐noEFR and EFR‐EFR will be comparable. Moreover, the ROM model is consistent with respect to the choice of δ and χ, which are the same as those used in the FOM model.In the EFR‐EFR, we apply a double stabilization. Specifically, we employ the EFR algorithm not only at the FOM level, but also at the ROM level. Indeed, after the POD modes are built from the EFR snapshots, we apply the EFR steps (I), (II), and (III) in a reduced setting, as specified in Algorithm e did in , we expaf 𝕌r aswr≐wr..(I)rwrnFor the sake of clarity, in Table Remark 2We remark that, for the NSE, the online phase still depends on the FOM dimension and this affects the EFR‐noEFR and EFR‐EFR performances in terms of computational time. For this reason we are not presenting a comparative analysis between the FOM and the ROM solutions with respect to the computational costs. To overcome this issue, hyper‐reduction techniques, such as the empirical interpolation method (EIM), may be employed, see, for example, References Algorithm 21nme6942-gra-1001-b3.40≤Re≤100. This benchmark has been thoroughly studied at full order levelIn this section, we analyze and compare the performances of EFR‐noEFR ∈ℝ2such that(x−0.2)2+(y−0.2)2−0.052=0}, which is depicted in Figure ν=10−3 and use no‐slip boundary conditions on ∂ΩDwall, representing the union of the lower and upper walls of the channel, and the cylinder wall . The time‐dependent inlet velocity leads to a Reynolds number that varies in time,0≤Re≤100. We perform our tests on a triangular mesh with hmin=4.46·10−3 and hmax=4.02·10−2 . We employ the Taylor‐Hood ℙ2−ℙ1 FE pair for velocity and pressure, respectively, and this leads to a FE space of dimension Nh≐Nhu+Nhp=14053.We consider the motion of an incompressible flow in a domain Ω≐{0,.2×=. After the POD procedure, we retain the first 2 modes for both velocity and pressure, representing 99.9% of the snapshot energy. Here, for the sake of notation, we use r≐ru=rs=rp=2. We show representative solutions of velocity for t=1 and pressure for t=4 in Figures  and , respectively. The relative log‐error temporal trend, displayed in Figure 10−3 for the velocity, and reducing the error by two orders of magnitude with respect to the EFR‐noEFR for both variables. The comparison between FOM and ROM aerodynamic coefficients over time is reported in Figure E‾CD=1.02, ÊCD=0.13, and E‾CL=1.69, ÊCL=0.11. The advantage of using the EFR‐EFR is remarkable, since we are reducing the relative L2‐errors of the force coefficients by an order of magnitude. Table We collect  −0.71,0.1, respec −0.71,0.1, respecExperiment 2. The next step in our comparison of EFR‐noEFR and EFR‐EFR is the numerical investigation of the EFR stabilization strategy both at the FOM level and at the ROM level. Specifically, we use χ=5·Δt=0.002 in the Relax step for both the EFR‐noEFR and the EFR‐EFR. This choice limits the amount of dissipation introduced by the DF in the Filter step of the EFR algorithm, and yields a more challenging test problem than Experiment 1 for both the EFR‐noEFR and the EFR‐EFR. Specifically, Experiments 1 and 2 share the same computational setting except that for Experiment 1 no relaxation is performed. Indeed, in Experiment 1 we use χ=1, and thusO(10−2) in the worst case scenario. Thus, the grad‐div stabilization is no longer needed and the FOM and ROM are consistent with respect to the grad‐div stabilization.nd thusurn+1=.We construct and test the EFR‐noEFR and EFR‐EFR on the time interval Nu=Np=2000 snapshots, which are equally spaced on the time interval . To retain 99.9% of the snapshots energy, we employ the following numbers of POD basis functions to build the ROMs: ru=43, rs=rp=8. We note that, with respect to Experiment 1, the system shows a slower decay of the eigenvalues and therefore more modes need to be used to construct the ROMs. The higher accuracy of EFR‐EFR is displayed in Figure CD(t) and CL(t) plotted in Figure L2‐errors of the force coefficients: E‾CL=0.26, ÊCL=0.11 and E‾CD=0.018, ÊCD=0.010. The maximum, minimum, and average error values over time for the velocity and pressure fields, which are listed in Table rp=rs=8 and choosing ru=30,32,34,36,38,40,42,44,46,48,50 shows that, over this range of ru values, the EFR‐EFR performs better than the EFR‐noEFR with respect to both the velocity and the pressure approximations. Indeed, the EFR‐EFR with ru=30 yields a low relative error that the EFR‐noEFR cannot attain by increasing its ru value .In order to approximate all the relevant features of the flow field, we increase the number of snapshots. Indeed, to build the EFR‐noEFR and EFR‐EFR, we collect terval 4,. To retaOverall, the numerical investigation in Experiment 2 yields the same conclusions as the numerical investigations in Experiment 1: the EFR‐EFR is more accurate than the EFR‐noEFR with respect to all the criteria used, that is, pointwise, average, maximum, and minimum velocity and pressure errors, lift and drag coefficient errors, and Pareto front. Thus, these numerical results suggest that the FOM‐ROM consistency is beneficial for the EFR stabilization strategy.Experiment 3. using Nu=Np=2000 snapshots that are equally spaced in this time interval. Thus, we are using coarser resolution than the resolution used in Experiment 2, since we are employing the same number of snapshots as in Experiment 2 but consider a time interval that is twice as long as that used in Experiment 2. Furthermore, in the first half of the time interval the flow displays laminar dynamics, whereas in the second half it displays more complex dynamics . We note that the mixed dynamics in Experiment 3 is more challenging to represent at the ROM level than the dynamics in Experiment 2 . In order to retain 99.9% of the energy of the snapshots, we choose ru=47 and rp=rs=7. We note that in Experiment 3 we utilize a similar number of basis functions as in Experiment 2. Since the dynamics in Experiment 3 is more challenging than the dynamics in Experiment 2, we conclude that the Experiment 3 ROM resolution is coarser than the Experiment 2 resolution.In this experiment, which is based on the computational setting in References Overall, in Experiment 3, neither EFR‐noEFR nor EFR‐EFR give satisfactory results. Furthermore, both EFR‐noEFR and EFR‐EFR are significantly less accurate in Experiment 3 than in Experiment 2. Indeed, the log‐relative errors for the velocity field reported in Figure t=8 displayed in Figure t∈ EFR‐EFR yields relative errors below 10−2 for the velocity field, this advantage is lost in the last part of the time interval, reaching unacceptable errors values (close to 1). On the other hand, concerning the pressure field  is well recovered only for the first part of the time interval, when the vortex shedding does not occur and the low frequency modes are dominant. Even the drag coefficient CD(t) is not reconstructed in a satisfactory way and shows spurious oscillations for t>4. This behavior is worse in the EFR‐noEFR results, which exhibit larger amplitude oscillations. The EFR‐noEFR and EFR‐EFR yield the following L2−errors of the force coefficients: E‾CL=2.39, ÊCL=1.2, E‾CD=0.33, and ÊCD=0.15. Overall, although the EFR‐EFR results are significantly more accurate than the EFR‐noEFR results, both ROMs yield relatively inaccurate results. These results show that, as expected, utilizing a more challenging regime  in Experiment 3 deteriorates the EFR‐noEFR and EFR‐EFR performance. We emphasize, however, that even in this more challenging regime EFR‐EFR performs better than EFR‐noEFR.This is confirmed by the velocity solution for 3.5χ and δ do not change with respect to the reconstructive setting although this choice may be suboptimal. In this section, we answer the following questions: (i) are the EFR‐noEFR and EFR‐EFR algorithms predictive? (ii) Which algorithm performs better in the predictive regime?This section focuses on preliminary results on the predictive capabilities of the EFR‐noEFR and EFR‐EFR algorithms. We stress that the offline phase and the parameters Experiment 1. To study the predictability of ERF‐EFR and EFR‐noEFR strategies, we collect Nu=Np=200 equally spaced snapshots for both velocity and pressure in . After the POD procedure, we retain the first 2 modes for both velocity and pressure since they represent 99.9% of the snapshot energy. We recall that the filter radius is δ=0.0032 and the relaxation parameter is χ=1. We test the predictive capability of the model in the time interval . In terms of relative velocity errors, EFR‐EFR performs better than EFR‐noROM, reaching values around 10−2 and reducing the error by two order of magnitude, as illustrated in the left plot of Figure t=9.4. After that value, the EFR‐noEFR error increases, while the EFR‐EFR remains stable around 10−1. For the sake of completeness, we report the L2‐error values over the force coefficients: E‾CL=0.23, ÊCL=0.08 and E‾CD=1.41, ÊCD=1.49. These values are consistent with Figure Experiment 2. The next step is represented by the analysis of the predictive regime in the setting of Experiment 2. Namely, we collect Nu=Np=2000 equally spaced snapshots in the time interval . We employ ru=43, rs=rp=8 to retain 99.9% of the snapshots energy. In this case δ=0.0032 and χ=0.002.T=11. We do not go further in time, since for t>11 the Newton's solver of the FOM simulation does not converge. From the plots in Figures L2−error over the force coefficients: E‾CL=1.00, ÊCL=1.00, and E‾CD=0.99, ÊCD=0.99. EFR‐EFR performs slightly better than EFR‐noEFR for all criteria, except for the drag coefficient. We note that, as in Experiment 3, both the EFR‐EFR and the EFR‐noEFR approaches struggle since the flow we investigate displays mixed dynamics .We analyze the predictive regime up to Overall, we conclude that (i) both the EFR‐EFR and the EFR‐noEFR approaches struggle in the predictive regime, and (ii) both approaches are comparable in terms of accuracy with respect to all the criteria. Finally, we also note that we do not investigate the EFR‐EFR and EFR‐noEFR algorithms in the predictive regime of the more challenging Experiment 3 since the two approaches struggled in the predictive regime of Experiment 2.4ν). To this end, we consider the NSE j,mp(νi)j and mS(p(νi))j, for j=1,…,Nt≐Nut=Npt=Nst. For the sake of simplicity, in our setting we choose the same number of modes for all the variables and for all the parameters in the training set. However, in principle, one can choose the number of modes for each parametric snapshot through energy criteria, and the number can be different for each variable.A global compression of the scaled modes. Using the POD procedure presented in Section To perform the model reduction both in the time domain and in the parameter domain, a standard POD approach that performs a simultaneous compression in time and in the parametric space would require a significant computational effort. Thus, to avoid the high computational cost of this brute force POD approach, in our numerical investigation we use a nested‐POD (n‐POD) algorithm. This compression algorithm is very popular (and goes by different names) in the ROM community: see, for example, References Nt denotes the first phase of the time evolution compression, while r and rp are the final reduced space dimensions for velocity and pressure, after supremizer stabilization.For the sake of readability, we introduce the following notation Pseudocodes that describe the EFR‐noEFR and EFR‐EFR approaches coupled with the n‐POD algorithm are reported in Algorithms Algorithm 31nme6942-gra-1002-bAlgorithm 41nme6942-gra-1003-bExperiment 4. In this experiment, we use the same test problem as the one used in Section ν∈, which yields Remax∈. To explain the rationale for choosing this parametric domain, we define the averaged Reynolds number asU‾ is the time averaged magnitude of the inflow velocity, uin. For our parametric domain, we choose ν=1.575×10−3 since this value yields , which is the lower bound of the kinematic viscosity that achieves a vortex shedding behavior in the case of a steady inlet condition . In Section Remax=100 .In Section 4.1t=8    Figure , left an) Figure , right. rp=rs=3 and choosing ru=20,22,24,26,28,30,32,34,36,38,40 shows that, over this range of ru values, the EFR‐EFR performs better than the EFR‐noEFR with respect to both the velocity and the pressure approximations. Indeed, the EFR‐EFR with ru=20 yields a low relative error that the EFR‐noEFR cannot attain by increasing its ru value .Overall, the improvement in the EFR‐EFR is highlighted by the left Pareto plot in Figure 4.2t=7    Figure , left. A) Figure , right, L2−errors of the force coefficients, EFR‐EFR is always more accurate than EFR‐noEFR. Indeed, E‾CL=5.55, ÊCL=4.08, E‾CD=0.04, and ÊCD=0.02. We also present a Pareto plot for Remax=100 in the right panel of Figure rp=rs=3 and choose ru=20,22,24,26,28,30,32,34,36,38,40. Over this range of ru values, we observe that, although EFR‐noEFR is optimal for smaller ru values, the relatively low EFR‐EFR error for ru=20 cannot be reached by EFR‐noEFR even by increasing its ru value .Focusing on Table Remax=65 and Remax=100 show that, for both Reynolds numbers and for all criteria, the EFR‐EFR is consistently more accurate than the EFR‐noEFR . Thus, the numerical investigation in this section suggests that the FOM‐ROM consistency is important when the EFR stabilization is used and model reduction is performed both in time and in the parametric domain.The numerical results for 4.3In this section, we analyze the predictive capabilities of the EFR‐EFR and EFR‐noEFR algorithms with respect to the Reynolds number. We note that we do not investigate the predictive capabilities of the two algorithms with respect to both time and Reynolds number since both algorithms struggled in the predictive regime for Experiment 2 in Section Remax=110. In our numerical investigation, we use the same computational setting as that used in Experiment 4. Specifically, we use δ=hmin and χ=0.002. We also collect Nu=Np=2000 equally spaced snapshots in the time interval  for Nν=20. For these snapshots, we choose a log‐equispaced distribution in the parametric domain ν∈. We employ ru=33 and rs=rp=3 to retain 99.9% of the snapshot energy. The relative error plots in Figure We first present results for L2−error of the force coefficients: E‾CL=4.77, ÊCL=3.99, and E‾CD=0.048, ÊCD=0.037.A similar behavior is observed with respect to the force coefficients, which are displayed in Figure Overall, these results yield the following conclusions: (i) the EFR‐EFR and EFR‐noEFR algorithms are predictive in the approximation of the velocity field and the drag coefficient, but both struggle in the approximation of the pressure field and the lift coefficient. (ii) The EFR‐EFR algorithm is consistently more accurate than the EFR‐noEFR algorithm with respect to all criteria, especially in the approximation of the velocity field.4.4Remax=140 and use the same computational setting as that used in Experiment 4. Specifically, we use δ=hmin and χ=0.002, and collect Nu=Np=2000 equally spaced snapshots in the time interval  for Nν=20. For these snapshots, we choose a log‐equispaced distribution in the parametric domain ν∈. We employ ru=33 and rs=rp=3 to retain 99.9% of the snapshot energy. The relative error plots in Figure L2−error of the force coefficients: E‾CL=3.91, ÊCL=3.64, and E‾CD=0.098, ÊCD=0.11. Overall, these results yield the following conclusions: (i) the EFR‐EFR and EFR‐noEFR algorithms are predictive in the approximation of the velocity field and the drag coefficient, but both struggle in the approximation of the pressure field and the lift coefficient. (ii) The EFR‐EFR and EFR‐noEFR algorithms perform similarly with respect to all criteria. We believe that, to increase the predictive capabilities of the EFR‐EFR and EFR‐noEFR algorithms, the parameters δ and χ should be tuned appropriately. This, however, goes beyond the scope of the current investigation.In this numerical investigation, we increase the Reynolds number to 5In this article, we took a step in the study of FOM‐ROM consistency when the EFR algorithm is used as numerical stabilization in convection‐dominated, marginally‐resolved flows. To this end, as a mathematical model we considered the incompressible Navier–Stokes equations. We used moderate Reynolds numbers, which yielded a convection‐dominated regime. We performed FOM and ROM simulations in the marginally‐resolved regime, that is, when the number of degrees of freedom is barely capable of capturing the main features of the underlying flow. To tackle the inaccuracies of the FOM and ROM simulations in the marginally‐resolved regime, we employed the EFR algorithm, which leverages spatial filtering to alleviate the spurious oscillations.the EFR‐noEFR, in which the EFR regularization is used at a FOM level, but not at a ROM level;the EFR‐EFR, in which the EFR regularization is used both at a FOM and at a ROM level.To investigate the FOM‐ROM consistency, we considered two models:Re=100. As criteria for our comparison, we used the relative velocity error, the relative pressure error, and the lift and drag coefficients. We also considered two types of model reduction: (i) model reduction in time, for which we used the POD algorithm; and (ii) model reduction in time and in the parameter space, for which we used the nested‐POD algorithm. In all our tests, for both types of model reduction, and for all three criteria, the EFR‐EFR was more accurate than the EFR‐noEFR. These results suggest that FOM–ROM consistency is beneficial for the EFR regularization in a convection‐dominated, marginally‐resolved regime.We investigated the EFR‐noEFR and EFR‐EFR in the numerical simulation of a 2D flow past a circular cylinder at time‐dependent Reynolds numbers with a maximum value under‐resolved regime, which is important in many realistic settings  where the ROM dimension is significantly lower than the number of degrees of freedom needed to accurately represent the complex dynamics of the underlying system. Related to this investigation, higher Reynolds number flows should be considered. Another important research direction is the investigation of FOM‐ROM consistency when different regularized models are used at the FOM and ROM levels . Related to this, one could also investigate the parameter FOM‐ROM consistency, which is complementary to the model FOM‐ROM consistency investigated in this article. Specifically, one could consider the same regularized model at the FOM and ROM levels, but use different parameters  in these regularized models. Using different δ or χ values at the FOM and ROM levels  could yield more accurate ROM solutions in several settings, such as hyper‐reduction.These first steps in the study of the FOM‐ROM consistency of regularized models are encouraging. There are, however, other research directions that should be investigated for a deeper understanding of this important topic. Probably the most important investigation should focus on the FOM‐ROM consistency for regularized models in the Finally, we emphasize that most of the existing studies (including this article) on the FOM‐ROM consistency have been numerical investigations. Although theoretical investigations could support the existing numerical investigations and shed new light on the FOM‐ROM consistency, these studies are relatively scarce (for notable examples, see the numerical analysis performed in References These numerical and theoretical investigations of various types of FOM‐ROM consistency could provide a new impetus for the development of ROMs that are consistent with their corresponding FOMs.


However, Ca-alginate shows low bioactivity and requires functionalization, increasing labor work and costs. In contrast, films of sodium alginate crosslinked with iron chloride  have shown good bioactivity with fibroblasts, but MSCs studies are lacking. We propose a first proof-of-concept study of Fe-alginate beads supporting MSCs proliferation without functionalization. Macro- and micro-carriers were prepared (extrusion and electrospray) and we report for the first time Fe-alginate electrospraying optimization. FTIR spectra, stability with various mannuronic acids/guluronic acids (M/G) ratios and size distribution were analyzed before performing cell culture. After confirming literature results on films with human MSCs, we showed that Macro-Fe-alginate beads offered a better environment for MSCs adhesion than Ca-alginate. We concluded that Fe-alginate beads showed great potential as ready-to-use carriers.Micro-carriers, thanks to high surface/volume ratio, are widely studied as mesenchymal stem cell (MSCs) in vitro substrate for proliferation at clinical rate. In particular, Ca-alginate-based biomaterials (sodium alginate crosslinked with CaCl Cell expansion at the clinical scale is a critical need to offer fast, reliable and cost-effective cell therapies for a large variety of diseases ,2. DiffeAmong various structures and materials, polymer beads have shown increasing interest as in vitro cell culture substrates, in particular due to the extremely high surface/volume ratio they offer compared to flat surfaces ,6,7,8,9.®, and a broad range of polymers and composites are reported in the literature for the preparation of homemade beads, such as polycaprolactone [2+ from Calcium Chloride (CaCl2) dissolved in water to form calcium alginate  beads with high throughput using various fabrication methods [Commercial microcarriers are available, such as Cytodexolactone  or alginolactone . It is solactone . It can  methods ,17,18. U methods . Blends  methods , cellulo methods , calcium methods  or fibro methods , are the3), approved by the Food and Drug Administration FDA for food industry [However, sodium alginate can be crosslinked with other ion sources to form scaffolds with alternative biological and physicochemical properties, such as iron chloride  of Fe-alginate beads with Ca-alginate ones and 2D culture surfaces as controls.After confirmation of the potential of Fe-alginate films as reported in literature, we (1) analyzed the effect of the alginate composition (M-to-G chain ratio), (2) optimized for the first time the fabrication of micro-sized Fe-alginate beads through electrospray and (3) assessed the early cell response of MSCs cultured on the bead surface. The hypothesis of this proof-of-concept study was that alginate solution crosslinked with iron chloride would be promising to form ready-to-use stem cell carriers  that could decrease costs and labor time for cell expansion systems.2) and iron chloride (FeCl3) were purchased from Sigma-Aldrich (USA). Information regarding the M/G ratio were provided by the manufacturer.Sodium alginate powders with low G-chain content  and high G-chain content , calcium chloride  solution, respectively. Iron chloride crosslinker solution at 1% was prepared by dissolving 100 mg in 10 mL of demineralized water. Calcium chloride crosslinker solution at 102 mM was prepared by dissolving 170 mg in 20 mL of demineralized water.A homemade electrospray set-up  was usedFor the fabrication of sterile beads in order to perform biological analysis, sodium alginate was dissolved in sterile water upon heating at 70 °C for 30 min (pasteurization). Crosslinker solutions were sterilized by filtering them through 0.22-µm syringe filters. The electrospray set-up and all tubing were cleaned with 70-% ethanol and washed thoroughly with sterile demineralized water. All glassware was autoclaved and beads were washed with sterile PBS in a biosafety cabinet (sterile laminar air flow) immediately after crosslinking. All steps were performed in a cell culture platform with restricted access.Optimal parameters  for the fabrication of micro- and macro-sized beads are reported in The same solutions  were used for the fabrication of alginate films as described by Machida-Sano et al. . The algPhase contrast images were acquired using an Eclipse Ti  microscope, and bead diameters were measured with the software NIS-Elements  using the “intensity profile” tool. Briefly, a linear section is drawn on the images through the center of a bead and the corresponding intensity histogram is plotted, allowing for the measurement of the diameter (length between the intensity shifts).The Fourier transform infrared spectroscopy (FTIR) spectra were acquired from raw sodium alginate powders and from crosslinked beads using an FTIR spectrometer  equipped with attenuated total reflectance .To assess the stability of the beads over time, Fe-Macro samples prepared with the different sodium alginate references were maintained in 12-well plates in 2 mL of PBS or culture medium  for one week at 4 °C or 37 °C. Ca-Macro beads were used as control. Pictures of the macroscopic behavior of the beads were acquired at day 7 with a Fujifilm XQ1 camera (Japan).2 in humidified atmosphere. Cells were then harvested at P10 using trypsin-EDTA  to be seeded on alginate films and beads.Green Fluorescence Protein (GFP)-producing human MSCs were kindly provided by the Department of Paediatrics and Adolescent Medicine, LKS Faculty of Medicine, The University of Hong Kong. MSCs were precultured in T75 flasks in DMEM culture medium , supplemented with 10% fetal bovine serum  and 1% penicillin-streptomycin  at 37 °C, 5% COOn films, cells were seeded in the wells of the 24-well plate hosting the films . Cells were also seeded in empty wells as plastic surface controls. Medium was refreshed every 2–3 days for one week .®  to prevent cells and micro-sized beads from adhering on the walls. Alginate beads were then pre-incubated in complete medium for 48 h in the vials prior to seeding. On macro-sized beads, culture medium was then removed and MSCs were seeded at an initial density of 1 million cells per vial in 500 µL. After two hours, fresh culture medium was added up to 5 mL per vial. On micro-sized beads, medium was refreshed (5 mL) and the same number of MSCs were injected in the beads suspension. Samples were then gently shaken every 15 min for 2 h to homogenize cell seeding.To culture MSCs on beads, glass bijou vials were coated with SigmacoteThe GFP-producing cells were observed with fluorescence microscopy using an Eclipse Ti  microscope. Images were acquired after one week (culture on films) and over time . Phase contrast images were also recorded to visualize beads more clearly.Metabolic activity of cells alone or with Fe-alginate beads  was evaluated with the Alamar Blue assay at days 1, 4 and 7 to evaluate the cytotoxicity of released ions. Briefly, the culture medium was replaced by fresh medium supplemented with 10% of Alamar Blue solution  and samples were incubated for 2 h under normal culture conditions. The background controls used as blank for the different conditions were, respectively, (1) Alamar Blue solution incubated alone  or (2) Alamar Blue in contact with beads only (no cells). The Alamar Blue solutions were then removed from the samples and the fluorescence intensity recorded at 560/595 nm with a spectrofluorimeter . Samples were put back in incubation with fresh culture medium. A high fluorescence intensity highlights important reduction of the dye by mitochondria and therefore high metabolic activity of the samples.2) for one additional week. Cells were then harvested from the flask surface with Trypsin-EDTA to check cell viability again with Trypan Blue. Fresh MSCs that had never been cultured on beads were seeded, simultaneously, in flasks with the same density as controls.After one week, cells were harvested using trypsin-EDTA and isolated from the beads with 70-µm cell strainers. Cell count and viability were assessed using Trypan Blue (1:1) and an automated cell counter . To evaluate cell recovery after expansion on beads, the same process was applied and cells were then put back in 2D culture in T75 flasks in normal culture conditions . All results were tested for statistical significance, the absence of specific notation in the figures corresponds to the absence of statistical significance. When the absence of significance is highlighted, it appears as “N.S.” in the figures.Quantitative data were plotted as mean and standard error of 3 independent experiments. Statistical significance was tested using non-parametric analysis of variance (no assumption of Gaussian distribution). Results were considered statistically significant for Results previously reported in the literature for fibroblast culture on Fe-alginate films were first repeated to confirm the potential of scaffolds based on alginate crosslinked with iron ions for enhanced cell bioactivity. Initially, Machida-Sano et al. showed that fibroblasts seeded on Fe-alginate films benefited from an appropriate substrate for cell adhesion and development, in contrast with Ca-alginate . Cell nuCulture of MSCs on Fe- and Ca-alginate films allowed us to confirm this trend with another cell source for the first time . SamplesThe study of alginate beads macroscopic behavior, namely swelling and degradation in an aqueous environment, was performed on macro-sized beads crosslinked with either iron or calcium chloride. Beads were immersed in either PBS or DMEM and kept in incubation at either 4 or 37 °C for one week.The chemical structure of sodium alginate is a combination of two different chains, guluronic acid (G) and mannuronic acid (M) . The algHere, initially, the use of alginate with a high M/G ratio  led to a very poor stability of the Fe-alginate beads in certain conditions, in particular DMEM at 37 °C . A very FTIR analysis was then conducted to evaluate crosslinking and confirm the difference in the M/G ratio between alginate references. The spectra of raw alginate powders and Fe-alginate beads are reported in −1 shifted to lower and higher values, respectively [Overall, the shifts occurring between the spectra of raw sodium alginate ,40,41 anectively ,42. Thisectively  and was ectively ,44. Diffectively .Therefore, we showed here the influence of the M/G ratio on bead stability and the successful crosslinking of sodium alginate with iron ions. High G-chain content sodium alginate was then used for the rest of the experiments reported in the present paper. It has to be mentioned that swelling still appeared on beads made from high-G sodium alginate, for both iron- and calcium-crosslinked samples. This did not jeopardize cell attachment as long as the swelling was over before cell seeding , which cThe fabrication of macro-sized beads requires only a stable flow rate of alginate solution dropped into a crosslinker bath , while dIn particular, electrospraying requires strict control of a number of process variables. Mostly, these variables are selected based on empirical knowledge for optimal bead formation. Maintaining the sphericity of the alginate beads is important for good mechanical and chemical stability. It was reported that the gel bead strength of non-spherical beads was greatly reduced as compared to spherical beads . In addip < 0.001). More importantly, with a low flow rate, a non-homogeneous, two-peak distribution appeared (p < 0.0001), and size distribution was improved with a smaller deviation for a higher voltage. These variations in diameter were consistent with results reported in the literature for calcium alginate particles [Results of bead diameter mean and distribution for various flow rates  and voltages  are reported in appeared A, while appeared C,D. The articles .The batch-to-batch reliability was then checked by comparing the size distribution of independent productions of beads, as seen in The complete size distribution analysis among the different groups  is reported in We first investigated the possible cytotoxic effects of Fe-alginate beads present in the culture medium on MSCs attached and grown on tissue culture plastic surface (6-well plate), to analyze if the release of specific ions in the environment could alter cell proliferation and viability over time. MSCs were cultured for one week with or without beads dropped in the culture medium. Metabolic activity was evaluated with Alamar Blue over time, then cells were harvested to measure cell number and cell viability at day 7. Results are reported in Both groups, with or without beads, showed an increased metabolic activity over time, highlighting cell expansion A. AssumiMSCs were therefore cultured directly on the surface of the beads for one week on Fe-alginate and Ca-alginate of both sizes. Phase contrast A and fluFrom the perspective of cell therapy or tissue engineering, cell expansion on carriers is just a single step in the process and the cells of interest have to be later harvested to be used for a specific application. Therefore, we analyzed the recovery potential of MSCs after growth and harvest from Fe-alginate beads. We were not able to harvest enough cells from Ca-alginate beads (macro or micro) to perform such a study on these groups. After one week, MSCs were detached from the surface of Fe-Micro and Fe-Macro samples with trypsin-EDTA and seeded in regular cell culture flasks. After one additional week of culture in a flask, cell viability was measured and compared to fresh MSCs that had been maintained in 2D culture as control . All celWe reported here for the first time the investigation of iron-crosslinked alginate beads as carriers for in vitro mesenchymal stem cell expansion. After optimization and characterization of the biomaterial, in particular stability through analysis of the M/G alginate chains ratio, we showed the higher bioactivity potential of Fe-alginate carriers compared to the well-known Ca-alginate scaffolds, leading to an increase in MSCs attachment and growth. These results validated our hypothesis that alginate crosslinked with iron chloride to form beads is of primary interest towards the development of cheap and ready-to-use cell carriers, compared to Ca-alginate products requiring additional functionalization steps to ensure relevant cell behavior. This study allowed us to show for the first time the production of Fe-alginate beads through electrospray, and to propose macro-sized beads as optimal parameters. Following this proof-of-concept approach, further studies could now investigate long-term use of this biomaterial, regarding cell behavior as well as degradation rate and evolution of mechanical properties. In particular, stemness markers could be investigated to ensure that MSCs remain undifferentiated even after a long culture period. The potential with other cell sources, in particular primary human MSCs, should now be considered, as well as the direct use in bioreactor systems. The outlooks also include the encapsulation of factors and drugs within the beads, to control the in vitro cell development but also for implantable drug delivery systems comprising both cells and beads for the filling of defects.