In response to incoming neuronal signals, each active zone protein plays a role in the release-ready site replenishment with synaptic vesicles for sustainable synaptic transmission. The active zone release apparatus provides a possible link between neuronal activity and plasticity. This review summarizes the mostly physiological role of active zone protein interactions that control synaptic strength, presynaptic short-term plasticity, and homeostatic synaptic plasticity.An action potential triggers neurotransmitter release from synaptic vesicles docking to a specialized release site of the presynaptic plasma membrane, the active zone. The active zone is a highly organized structure with proteins that serves as a platform for synaptic vesicle exocytosis, mediated by SNAREs complex and Ca Presynaptic action potential (AP) triggers this process by opening voltage-gated Caproteins ,2. Ca2+ eceptors ,2. The neceptors ,2,3 that2+ dynamics-dependent replenishment of the neurotransmitter release site with release-ready SVs that involves multiple protein cascades, such as calmodulin-binding [This review introduces, at first, recent findings on temporal regulation of SV states within 100 ms of AP , current-binding  and phos-binding . These p-binding ,12,13 an-binding ,15,16. TV channels [Synapses are intercellular junctions between a presynaptic neuron and a postsynaptic neuron—precisely opposed pre- and post-synaptic specializations that contain electron-dense material on their plasma membranes . Ultrastchannels . This AZ2+ sensor, and Munc13, an AZ protein, still engaged [2+-dependent and temporary and lasts for 100 ms or less. Newly docked vesicles undock or fuse within 100 ms, suggesting that the sequence of rapid redocking, and subsequent slow undocking, may underlie the facilitation of synaptic transmission. These snapshot images at defined time points demonstrated time-dependent millisecond SV dynamics, synchronous and asynchronous fusion, undocking, and docking, controlled by transient Ca2+ elevation with opening of CaV channels with membrane depolarization accompanying AP.To characterize spatial and temporal organization of SV fusion sites following AP, the ‘zap-and-freeze’ method, generating an AP with a 1 ms depolarization pulse and following high-pressure freezing at defined time points, was recently developed by Watanabe and co-workers . Applyin engaged . During  captured evoked SV dynamics at 5 ms, showing that synchronous fusion of multiple SVs occurs within 5 ms throughout a single AZ. This observation is consistent with evoked synaptic currents with delay <1 ms and a typical rise time of 1–3 ms [.Watanabe and co-workersf 1–3 ms .2+-triggered fusion. Corresponding timescales with high-frequency synaptic activity and concomitantly increased Ca2+ concentrations can proceed within: step (1) 10–50 ms, step (2) 1–5 ms, and step (3) 0.2–1 ms. These timescales estimated by electrophysiological studies are similar to but a bit faster than those of morphological observations induced by an AP [2+ with high-frequency synaptic activity. Furthermore, a recent study proposes that vesicles transit sequentially from a loosely docked state to a tightly docked state before exocytosis, and Mover, an AZ protein, selectively controls the release probability of tight-state primed SVs in the mouse calyx of Held [.Dynamically primed SV states for buildup of the molecular machinery that mediates SV fusion have been summarized by Neher and Brose , as follby an AP , due to  of Held .V channels (b), RIM-BP (RIM-binding protein), Bassoon, Piccolo, Liprin-α, and CAST /ELKS ,30,31,32synapses . This laf the AZ .V channels at the release site [V2 channels [2+ channels [2+ channels, that determines recruitment of Ca2+ channels to the AZ, includes RIM-BP [2+ channel topography at a fast auditory pathway central synapse [V2 channels, disruption of AZ structure, and loss of vesicle docking, but not loss of Liprin-α [V channels in the AZ, while RIM, RIM-BP, and CAST/ELKS may be essential for constitution of the AZ structure.The AZ of hippocampal excitatory synapses contains several clusters of the scaffold RIM, and these clusters mark individual release sites . RIM medase site   and SVs from rat brains coat the surface of condensed liquid droplets formed by AZ proteins RIM, RIM-BP, and ELKS via phase separation [2+ channels anchored on membranes. These studies reconstitute a presynaptic bouton-like structure mimicking the SV-tethered AZ with its one side attached to the presynaptic membrane and the other side connected to the synapsin-clustered SV condensates  binding to the Munc13 C2B domain potentiate SV exocytosis, thereby offsetting synaptic depression induced by vesicle depletion [2+-independent action of RIM for control of release efficacy was also proposed, as follows: the Ca2+-independent interaction between RIM C2B domains and PIP2 coordinates the efficient function of fusion machinery by controlling the relative position of PIP2 and secretory proteins that depend on it [As described above, RIM, interacting with cytoplasmic C-terminal sequences of Cachannels ,38 and Schannels , may parepletion . A possind on it .2+ sensor and SNAREs fusion machinery, Munc18 accelerates SNARE proteins assembling through direct contact with both t- and v-SNAREs [2 and C1 domains [For SV fusion, before the actions of the Cav-SNAREs . Deletiov-SNAREs . Howeverv-SNAREs . Munc13- domains ,74 and d domains ,75,76. M domains . Munc13- domains . Thus, M2+-independent neurotransmitter release from SVs in glutamatergic hippocampal autaptic neurons. Compared with the control neurons, the RRP size was severely reduced by Munc13-1 deletion (~95%) or RIM1/2 deletion (~88%), suggesting the requirement of both RIM1/2 and Munc13-1 for efficient SV priming. In addition, the activity of both RIM1/2 and Munc13-1 is essential for AP- or Ca2+-evoked SV fusion in the hippocampal autaptic neuron. Although neurotransmitter release efficiency is controlled uniquely by RIM1/2, activity-dependent augmentation of SV pool size relies exclusively on the action of Munc13s. Rosenmund and his co-workers concluded that RIM and Munc13-1 act in overlapping and independent states of SV localization and release. It is likely that, in the resting state, RIM localizes SVs in the right position, in the proximity of the AZ membrane, and then RIM together with Munc13 forward the SV state to docking and priming on the AZ membrane [For the SV localization factor, the dynamic tripartite complex of α-RIMs, Munc13, and Rab3, an SV protein, is suggested to mediate SV targeting to the release sites . Their rmembrane .S45 in sympathetic presynaptic terminals was observed with APs burst (20 < 50 Hz) [S45D expression in the sympathetic presynaptic neuron reduced the peak amplitude and the integral of EPSP, and the SV number in the RRP [S45 phosphorylation reduces the number of release-ready SVs. The paired-AP protocol experiments indicate that phosphorylation of CASTS45 with the time window of 30–120 ms after the first AP induces paired-EPSP depression. Overexpression of the phosphonegative-CASTS45A-mutant reduces the paired-EPSP depression (<200 ms), suggesting that phosphorylated CASTS45 downregulates SV reloading shortly after AP, but not over longer (200 ms) time courses. This means that AP activates a kinase in the AZ to phosphorylate CASTS45 that negatively controls replenishment of the release site with release-ready SV. The possible kinase is an AZ-associated serine/threonine kinase SAD-B that phosphorylates CASTS45 in vitro [Immunocytochemical experimentally, an activity-dependent increase in phosphorylated CAST< 50 Hz) . The pho the RRP . Thus, tin vitro .Acute deletion of CAST by short-hairpin RNAs in sympathetic neuron synapses did not alter EPSP amplitude, however, it significantly delayed the rate of the fast reloading of release-ready SVs, following the RRP depletion with APs burst . Indeed,Bassoon mediates reloading of SVs to release sites at central excitatory synapses—cerebellar mossy fiber to granule cell synapses . BassoonV channels [Drosophila rbp null mutation proposed that RIM-BP action participates in a rate-limiting stage required for the replenishment of high release probability SVs following vesicle depletion [2+-dependent fast SV replenishment. Ultrastructural studies showed a reduction of docked and proximal SVs, in addition to an impaired topography of CaV channels in the AZ [V2.1 channels’ localization in the AZ [V2.1 channels, weaken P/Q-type Ca2+ current-driven synaptic transmission of cultured hippocampal neurons, and result in a higher relative contribution of neurotransmission dependent on CaV2.2 channels [2+-dependent fast SV replenishment.RIM-BP, binding to both RIM and Cachannels , is an eepletion . Recent epletion . RIM-BP2n the AZ . Bindingn the AZ . A genetchannels . It is lShort-term synaptic plasticity occurs during and after repetitive presynaptic AP activity on a timescale of milliseconds to minutes, and manifests in the modulation of synaptic efficacy via changes in the release probability and the RRP size .2+-dependent fast SV replenishment [RIM1, an essential protein forming droplet-like condensates which reconstitutes the SV-tethered AZ ,48 see , functionishment , suggest2+-dependent manner via an evolutionally conserved calmodulin recognition motif. Calmodulin binding to Munc13 proteins causes increased priming activity and the RRP sizes in autaptic hippocampal neurons. Thus, calmodulin and Munc13 form a Ca2+ sensor/effector complex and its activation by elevation of residual Ca2+ during and after repetitive AP shape short-term plasticity characteristics [2+-calmodulin-insensitive Munc13-1 exhibits a slower rate of SV replenishment, aberrant short-term depression, and reduced recovery from synaptic depression after high-frequency stimulation. Therefore, Ca2+-calmodulin/Munc13-1 complex is a pivotal component of the molecular machinery that determines short-term synaptic plasticity characteristics [2+ level activates calmodulin, which in turn liberates Munc18-1, causing short-term synaptic facilitation, such as paired-pulse facilitation [2+-calmodulin during and after repetitive AP to facilitate transmitter release.The neurotransmitter release probability is controlled by RIM, while activity-dependent augmentation of SV pool size relies on the action of Munc13 proteins  that funeristics . In miceeristics . Furtherlitation . In the litation . AZ protdownstream target in the PKC pathway. Expression of a PKC-insensitive Munc18-1 mutant in Munc18-1 null neurons does not show Diacylglycerol-induced potentiation and increased synaptic depression [Activation of Munc13 by phorbol esters is essential for synaptic potentiation . Proteinpression . Post-tepression . A recenpression . The calpression . These f2+-dependent activation of Bassoon and Piccolo for their regulation of the short-term depression has been reported, however, contribution of a kinase associated with the AZ cytomatrix is a possible candidate.Short-term depression during high AP activity, but not basal SV release, is controlled by Bassoon  and PiccS45 [S45 in vivo. Expression of phosphonegative-CASTS45A reduced the depression, while that of phosphomimetic-CASTS45D increased the depression of the second EPSP even at 2000 ms after the first AP [S45 phosphorylation potentiates paired-pulse depression, while dephosphorylation of CASTS45 reduces it. Therefore, phosphorylation of CASTS45, within 200 ms of AP firing, negatively controls rapid replenishment of the release site (seeSection 5) with SV docking , which modulates presynaptic membrane voltage and, thereby, controls Ca2+ channel activity for homeostatic neurotransmitter release, is required at the Drosophila neuromuscular junction [At the RRP size . In addiRRP size . This oc release ,92. Howe+ influx . Synapti+ influx ,92. In r blocked . Thus, djunction .Drosophila neuromuscular junction, RIM-BP is also essential for the homeostatic modulation of neurotransmitter release, being required for both the enhancement of Ca2+ influx and the enlargement of the RRP [2+ influx and the RRP size. Loss of RIM-BP slows the rate of high release probability SV resupply by ~4-fold at basal synaptic activity [2+ channels and RIM-BP in the AZ. Thus, RIM-BP is required for stabilized baseline neurotransmission, coupling SVs to sites of Ca2+ influx, and for the persistent homeostatic modulation of presynaptic neurotransmitter release through coordinated control of Ca2+ influx and dynamics of the high release probability SV pool [ is likely the primed SV in mammalian synapses (see ). Molecular perturbation studies in hippocampal neurons suggest that RIM-BPs promote SV priming by interaction with Munc13 [.At the  the RRP . In addiactivity . In contactivity . It is p SV pool . The higpses see . Moleculh Munc13 .SV replenishment of release sites at central excitatory synapses—cerebellar mossy fiber to granule cell synapse [calyx of the Held synapse during high-frequency synaptic activity [In a mammalian central nervous system, the fast central endbulb synapse of auditory nerve fibers to bushy cells of the cochlear nucleus, disruption of Bassoon slows SV replenishment and induces homeostatic plasticity . In the  synapse  and the activity . Thus, i2+ channels, with membrane depolarization accompanying AP [2+ dynamics that control SV states, synchronous and asynchronous fusion, undocking, redocking, and priming [2+ dynamics activates multiple protein cascades via Ca2+-sensor molecules [2+ dynamics, mediates the SV dynamics for regulation of neurotransmitter release probability, controlling the SVs/Ca2+ channel coupling. For this control, RIM and RIM-BP, required for clustering the Ca2+ channel at release sites [2+ dynamics also regulates the RRP size, controlling the SV states of docking and priming. CAST/ELKS and Munc13 are major key proteins for docking and priming, respectively [ELKS has differential, synapse-specific effects on the RRP and the release probability, although it is proposed that ELKS N-terminal domains have important roles in SV priming [.At the presynaptic terminal, SV fusion takes place within the sub-millisecond opening of nearby Canying AP . AP trig priming . The und priming . Our eleolecules ,94 to coolecules . The AZ se sites ,38,42, aectively ,61. Nota priming ., SV replenishment supports stable synaptic transmission. Bassoon, Piccolo, and Clarinet [Caenorhabditis elegans, play a role in SV tethering under sustained high AP activity to maintain stable synaptic transmission [During and after repetitive AP firingClarinet , a homolsmission ,49,59.2+-dependent regulation of the release probability via SV fusion machinery has been proposed for the generation of presynaptic plasticity [2+ channel clustering nearby the release apparatus [2+-dependent modulation of AZ protein function is mediated by calmodulin-binding [2+-independent modulation of AZ protein function also contributes to presynaptic plasticity [.Presynaptic plasticity is dependent on flexible neurotransmitter release. Presynaptic depression is due to reduced neurotransmitter release, while presynaptic facilitation or potentiation is due to increased neurotransmitter release. Residual Caasticity ,97. Howepparatus ,38,42. Ipparatus , that ofpparatus ,70, and pparatus ,49,59. C-binding ,85, phos-binding ,13 and S-binding , and dep-binding . Calmoduasticity ,81,87.Synaptic connections undergo homeostatic readjustment in response to changes of synaptic activity, to ensure a stable and flexible nervous system ,89,90,91


Avian influenza virus (AIV) subtypes H5 and H7, possessing the ability to mutate spontaneously from low pathogenic (LP) to highly pathogenic (HP) variants, are major concerns for enormous socio-economic losses in the poultry industry, as well as for fatal human infections. Through antigenic drift and shift, genetic reassortments of the genotypes pose serious threats of increased virulence and pathogenicity leading to potential pandemics. In this study, we isolated the H7-subtype AIVs circulating in the Republic of Korea during 2018–2019, and perform detailed molecular analysis to study their circulation, evolution, and possible emergence as a zoonotic threat. Phylogenetic and nucleotide sequence analyses of these isolates revealed their distribution into two distinct clusters, with the HA gene sharing the highest nucleotide identity with either the A/common teal/Shanghai/CM1216/2017, isolated from wild birds in Shanghai, China, or the A/duck/Shimane/2014, isolated from Japan. Mutations were found in HA (S138A (H3 numbering)), M1 (N30D and T215A), NS1 (P42S), PB2 (L89V), and PA (H266R and F277S) proteins—the mutations had previously been reported to be related to mammalian adaptation and changes in the virulence of AIVs. Taken together, the results firmly put forth the demand for routine surveillance of AIVs in wild birds to prevent possible pandemics arising from reassortant AIVs. Orthomyxoviridae, possess the ability to adapt to multiple hosts, including wildlife, domestic animals, and humans, causing yearly outbreaks with high rates of morbidity and fatality [Influenza A viruses (IAV), belonging to the family fatality . The virfatality . GeneticAmong the IAVs, H5 and H7 subtypes have been reported to undergo genetic mutations in their HA gene, and thereby transform from low pathogenic (LP) to high pathogenic (HP) ,4. Wild The Republic of Korea has a climate with long and dry winters, which seem to provide preferrable conditions for the continued evolution of influenza viruses by increasing the possibility of mixed genomes occurring between lineages within subtypes . TherefoIn this study, through national surveillance of IAVs, we isolated nine strains of H7N7 from wild birds during 2018, and four strains of H7N7 and one strain of H7N3 during 2019 in the Republic of Korea. To gain extensive knowledge about the evolution pattern of these viruses and the possible zoonotic threats thereby, we performed phylogenetic analysis based on each segment of the isolates, and compared them with other potent IAV sequences. In addition, we investigated the possible mutations affecting crossover from one species to another, and assessed the possible evolution of H7 IAV subtypes and their potential as a future zoonotic threat.During December 2018 and 2019, fresh fecal samples were collected from wild bird habitats early in the morning before sunrise. Among the 2019 isolates, KNU18-104 (A/wild duck/South Korea/KNU18-104/2018(H7N7)) and KNU18-113 (A/wild duck/South Korea/KNU2018-113/2018(H7N1)) samples were isolated from the feces of wild ducks, obtained nearby Geumho river, Gyeongsang province  and Wonju river, Wonju-si, Gangwon-do , respectively. KNU18-119 (A/White-fronted Goose/South Korea/KNU18-119/2018(H7N7)) samples were isolated from the feces of white fronted geese, obtained nearby Sihwa lake, Hwaseong-si, Gyeongii-do . KNU18-106~110 (A/wild duck/South Korea/2018(H7N7)) were isolated from the feces of wild ducks, obtained nearby Namdae stream, Yangyang-gun, Gangwon province. Among the 2019 isolates, KNU2019-14 /South Korea/KNU2019-14/2019(H7N7)) was isolated from the feces of mallards, obtained nearby Gunpyeong-ri, Osong-eup, Chungbuk province , KNU2019-25 ) from Sangrim-ri, Jillyang-eup, Gyeongsangbuk province , KNU2019-30 ) from Sin-dong, Chuncheon, Gangwon province , KNU2019-33 ) from Gahyeon-dong, Weonju, Gangwon province , and KNU2019-39 (A/White fronted Goose/South Korea/KNU2019-39/2019(H7N7)) from the feces of mallards obtained nearby Songchon-dong, Paju-si, Gyeonggi province . The fecal samples were transported in viral transport medium (VTM) supplemented with antibiotics. Two hundred microliters of VTM containing the fecal samples were inoculated into the allantoic cavities of 9–11-day-old specific-pathogen-free (SPF) embryonated eggs. The eggs were then incubated at 37 °C, and embryonic status was checked every day thereafter. After 5 days of incubation, the eggs were chilled, and allantoic fluids were subsequently harvested and tested for the presence of viruses by estimating hemagglutination activity using chicken erythrocytes, or by genetic confirmation through qRT-PCR using gene-specific primers. For host species determination, the mitochondrial gene and cytochrome c oxidase I (COI) gene were amplified with specific primers.A NucleoSpin RNA kit  was used to extract viral RNA directly from the allantoic fluid of embryonated chicken eggs (ECEs) according to the manufacturer’s instructions. In a nutshell, virus-containing egg fluid was combined with β-mercaptoethanol-containing lysis buffer. Filtration through a NucleoSpin filter lowered the suspension’s viscosity, and ethanol was added to alter the RNA-binding conditions. Desalting was accomplished by adding a membrane desalting solution to solubilized RNA attached to a NucleoSpin RNA column membrane. Because rDNase treatment could potentially reduce RNA stability, the DNA digestion step was skipped. After washing the desalted membrane with wash buffers, elution was conducted immediately. The first-strand cDNA was transcribed using a Superscript III first-strand cDNA synthesis kit  in a final volume of 20 µL, according to the manufacturer’s instructions, using a universal primer for influenza A virus (Uni12: 5′-AGCRAAAGCAGG-3′).NGS was conducted by GnCBio  following the HiseqX method, as reported previously . The cDNhttps://platform.gisaid.org/epi3/frontend#22c194) and NCBI, were downloaded on 31 May 2021. Each nucleotide sequence of the isolated subtype virus was queried against the downloaded database using nucleotide BLAST (NCBI) with default parameters, and sequences of the top 100 hits were collected. Thus, for each gene, 800 similar sequences were obtained, and sequences with the same strain name were removed. Phylogenetic analysis was performed for two rounds using MUSCLE and the maximum-likelihood method implemented in MEGA v.6.017. In the first round, 200 bootstrap replicates were run for all sequences. Based on the result of first round, in the second round, we selected several representative reference sequences, and pulsed H7N7 Korea strain sequences, forming smaller data sets. Phylogenetic analysis was repeated using the method described above with bootstrap 1000.In this study, the gene sequences of influenza A virus, available from the GISAID  approach, sampling every 5000 steps to obtain a post burn-in effective sample size (ESS) of at least 200 in all parameters. The maximum clade credibility tree was obtained by using Tree Annotator v1.10.4 with 10% burn-in. The final MCC tree was visualized using FigTree v1.4.4.Nucleotide sequences were deposited in the GenBank with the accession numbers mentioned in During 2018–2019, we isolated a total of 14 H7 isolates from wild bird habitats in the Republic of Korea, out of which, nine were isolated in 2018, and five in 2019. Genome sequence information of the isolates was deposited in GenBank with the accession numbers mentioned above. GenBank accession numbers of the eight gene segments and the highest nucleotide identities from the GenBank database are shown in In the isolate KNU2019-14 from 2019, surface gene HA and polymerase basic protein (PB)1 were closely related to A/common teal/Shanghai/CM1216/2017 (H7N7), which originated from China, whereas NA was closely related to A/mallard duck/Georgia/10/2016 (H7N7), with nucleotide identities of 99.51%, 99.64%, and 97.88%, respectively—the HA, PB1, and NA segments of another isolate KNU2019-33 were closely related to A/common teal/Shanghai/CM1216/2017 (H7N7). Most of the H7N7 isolates had homology associated with the isolates from China, except for one H7N3 isolate KNU2019-25, where HA was closely related to A/Jiangsu/1/2018 (H7N4). However, PB1 and NP were closely associated with sequences from the isolates obtained from ducks in Cambodia, namely H10N7 and H7N4, respectively.The 14 H7 isolates were subjected to molecular characterization . The molhttp://www.gisaid.org) on 31 May 2021  with A/duck/Assam/DUOR1512100028/2015 (A/H3N8) predict inheritance of the NP gene, and that of the MP gene through ducks from Bangladesh (A/duck/Bangladesh/26980/2015 (A/H7N9)) and Fukui, Japan. The NS gene from A/teal/Egypt/2016 got reassorted into a duck in Hunan with H6N6, and further transferred to our isolates of category 1. The PB2/PA were predicted to be transmitted from isolates in Mongolia with H7N3 in 2017. All the isolates had Eurasian lineage.In cluster 2, involving most of our isolates from 2019 and a few from 2018, such as KNU114, KNU113, and KNU119, most of the segments were inherited from the reassorted viruses of 2017, the Korean isolates of 2016 and 2017, and from isolates in Japan from 2014–2016. The 2017 reassorted viruses possessed PB2 from Netherlands/2015/H3N8, NP/MP from A/teal/Chany/2017/H12N5-like viruses, NP/MP and NS from AIVs isolated from Egypt/2015-2016, HA/NA from A/mallard duck/Georgia/2016/H7N7, and PB2/PB1/PA from Mongolia/2015/2017. Detailed information on the evolutionary reassortment is presented in The major force guiding the evolution of AIVs is the adjustment with new hosts or the escape from the pre-existing host community . This prWe identified 26 changes in the human and similar avian H7 viruses’ genomes that could be involved in human adaptation or increased viral replication. Mutation in the HA segment may produce structural changes in the protein, affecting receptor binding affinity. All of the H7 viruses tested lacked Q226L and S138A  in the PFrom our analysis, it is predicted that in 2018–2019, two different subgroups of H7 viruses might have been introduced into the Republic of Korea via different migratory bird populations through fecal contamination. The locations of circumpolar arctic and subarctic breeding grounds of migrating birds flying over numerous flyways in Eurasia overlap, with some sharing common breeding areas . Thus, sOn the basis of available resources, we proposed an evolutionary mechanism that could have led to the generation of novel H7 LPIAVs. The first phase of reassortment might have occurred among the cluster 1 viruses and other LP AIVs circulating in the Eurasian migratory bird pathway from 2015 to 2017. In the second phase, all the accumulated reassortments in the H7 viruses isolated between 2015–2016 from the Republic of Korea , togetheIn our proposed cluster 2 H7 reassorted viruses, the reassortments may have occurred through multiple phases. From phylodynamic analysis, it seemed that some H7 viruses of 2018–2019 had reemerged from the circulating viruses of 2011–2015. The PB2 segment from Netherlands/2015/H3N8, HA/NA from A/mallard/Georgia/2016/H7N7, PA from Bangladesh, and NP/MP from A/teal/Chany/H12N5-like viruses might have reassorted initially, and led to the evolution of a group of reassorted H7 viruses in 2017, which, further with segments from Korean isolates of 2016–2017 and isolates from Japan, might have led to the cluster 2 of H7 reassorted viruses of 2018–2019 in Korea. Thus, novel H7 reassortant viruses could have undergone further genetic reassortment with other LP IAVs, and spread across migratory bird species in these breeding sites prior to their subsequent dissemination during southern migration in 2016.In summary, the study provided valuable information about the geographical distribution, representation, genetic diversity, and possible evolutionary pathways of novel H7 reassortants in wild bird habitats of the Republic of Korea during 2018–2019. H7 viruses in both the clusters with considerable genetic diversity were detected at an unusually high frequency in the winter of 2018–2019. The efficient subclinical replication and shedding of viruses by chickens might facilitate their silent spread among poultry. Phylogenetic and nucleotide sequence analyses of these isolates from the Republic of Korea revealed their distribution into two distinct clusters, with mutations that have been previously reported to be related to mammalian adaptation and changes in the virulence of AIVs. Thus, the recent appearance of novel H7 reassortants in migratory bird habitats highlighted the need for continued influenza surveillance in both wild birds and poultry in the Republic of Korea to prevent future pandemics, as well as huge economic losses.


The impact of COVID-19 virus infection during pregnancy is still unclear. This systematic review and meta-analysis aimed to quantitatively pool the evidence on impact of COVID-19 infection on perinatal outcomes. Databases of Medline, Embase, and Cochrane library were searched using the keywords related to COVID-19 and perinatal outcomes from December 2019 to 30 June 2021. Observational studies comparing the perinatal outcomes of COVID-19 infection in pregnancy with a non-infected comparator were included. The screening process and quality assessment of the included studies were performed independently by two reviewers. Meta-analyses were used to pool the comparative dichotomous data on perinatal outcomes. The database search yielded 4049 results, 1254 of which were duplicates. We included a total of 21 observational studies that assessed the adverse perinatal outcomes with COVID-19 infection. The odds of maternal death (pooled OR: 7.05 [2.41−20.65]), preeclampsia (pooled OR: 1.39 [1.29−1.50]), cesarean delivery (pooled OR: 1.67 [1.29−2.15]), fetal distress (pooled OR: 1.66 [1.35−2.05]), preterm birth (pooled OR: 1.86 [1.34−2.58]), low birth weight (pooled OR: 1.69 [1.35−2.11]), stillbirth (pooled OR: 1.46 [1.16−1.85]), 5th minute Apgar score of less than 7 (pooled OR: 1.44 [1.11−1.86]) and admissions to neonatal intensive care unit (pooled OR: 2.12 [1.36−3.32]) were higher among COVID-19 infected pregnant women compared to non-infected pregnant women. Coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus, continues to be an alarming global public health crisis . This siWe conducted this systematic review and meta-analysis based on the PROSPERO protocol registered on 18 May 2021 CRD42021254974). This review included studies focused on perinatal outcomes of COVID-19 infection in pregnancy, mainly to evaluate the reported adverse perinatal outcomes in COVID 19 infected mothers and the prevalence of adverse perinatal  outcomes in COVID-19 infected pregnant women. This review reports adverse maternal, fetal, and newborn outcomes of COVID-19 infected pregnant women, comorbidities in COVID-19 infected pregnant women, and the incidence of COVID-19 infection among pregnant women in line with the updated PRISMA 2020 guidelines for reporting systematic reviews [54974. ThObservational studies (cohort and case–control) investigating the perinatal outcomes in COVID-19 infected pregnant women and published as peer-reviewed articles in English were eligible for inclusion. Case reports, case series, editorials, letters to the editor, perspectives, conference papers, narrative or systematic reviews, and studies without a non-infected pregnant group as the comparator were excluded. We searched Medline, EMBASE, and Cochrane Library databases to identify the published studies from December 2019 to 30 June 2021. The search strategy included a combination of keywords for COVID-19 and perinatal outcomes .All of the identified studies from the database search were exported to EndNote reference management software (version EndNote X9.3.3.). Then, Covidence systematic review software  was used to manage the independent screening process at both the stages of title and abstract screening  and full-text screening . Reasons for full-text exclusion were documented at the full-text screening stage. Any disagreement was resolved by consensus or by consultation with a third reviewer at both stages.The data from the included studies were extracted to an Excel sheet by one author, and another author cross-checked the accuracy. The extracted data included the study characteristics , participants’ socio-demographic and baseline data, comorbidities, adverse perinatal outcomes , preterm pre-labor rupture of membrane (PPROM), intrauterine death, fetal distress, preterm birth, low birth weight, stillbirth, Apgar score, admissions to neonatal intensive care unit (NICU), neonatal deaths, cesarean section deliveries, and operative vaginal births, the incidence of COVID-19 among pregnant women, and the outcome of interest of each study.Quality assessments of the included studies were performed using the National Institute of Health’s (NIH) study quality assessment tool for observational, cohort, and cross-sectional studies and the NIH study quality assessment tool for case–control studies . The qua2 statistic, where substantial heterogeneity was defined as I2 ≥ 30. Random effects estimates of the pooled odds of each perinatal outcome and comorbidity condition were generated using the Mantel–Haenszel method. The findings of each outcome comparison were summarized with odds ratio, 95% confidence interval, p-value, and the I2 statistic. Funnel plots were generated to visually evaluate the presence of publication bias.The characteristics of the included studies, characteristics of the COVID-19 infected pregnant women and the summary findings were tabulated. Further, the incidence of COVID-19 among pregnant women was graphically presented. Quantitative meta-analysis was carried out to pool the comparative dichotomous data of perinatal outcomes when more than one study presented the data for the relevant outcome. If individual studies reported no adverse outcome in the infected group or non-infected group, they were excluded from the meta-analysis of that particular perinatal outcome. Heterogeneity of studies was determined using the ITwo thousand seven hundred ninety-five 2795) studies were identified through the search engines for the title and abstract reviews after removing 1254 duplicates. Out of the total screened abstracts, 120 were selected for full-text screening, of which 99 studies were excluded (mainly due to lack of a comparison group or the presentation of inadequate data), and 21 95 studie,34,35,36There were nine (42.9%) articles from single-center studies ,22,25,27Regarding the quality of the included cohort studies, 10 criteria out of 14 (71%) were satisfied by 40% of the included studies. Almost all of the studies had clearly stated research objectives, clearly defined study populations, clearly defined valid and reliable outcomes, and over ≥50% participation rate by eligible persons. In almost all the included studies, the quality assessment was unable to determine the level of exposures related to examined outcomes, exposure measures more than once over time, and follow-up after baseline. Blinding of the assessors to the exposure status was a serious concern for all the included studies. Only 40–55% of the included cohort studies were marked positively for the criteria of adjusting for potential confounding factors and having a justified sample size. With regards to the quality of included case–control studies, eight criteria out of 12 (75%) were satisfied by 60% of the included studies. All the studies satisfied the criteria related to clearly defined objective/s, clearly defined study population, selection of the control from the same population, consistent use of defined inclusion and exclusion criteria, clearly defined and differentiated case and control groups, ability to confirm the exposure occurred prior to the development of the condition, implementation of valid and reliable exposure measures, and measuring and adjusting for confounding variables. Blinding of the assessors to the exposure status was not determinable in all the studies. Less than 35% of the included case–control studies had a justified sample size A,B. IndiEleven studies reported the incidence of COVID-19 among pregnant women, with rates ranging from 1.3% to 27%. Only cohort studies were used to determine the incidence of COVID-19 infection in pregnant women. Even though there were 18 cohort studies, a few did not report the total number of admissions, making it difficult to quantify the incidence. Among the 11 that reported incidence, there were six studies from the USA ,25,29,31In the 21 included studies, a total of 14,131 COVID-19 infected pregnant women were studied compared to 585,376 COVID-19 non-infected pregnant women. The reported mean age of infected pregnant women ranged from 24.7 to 32.6 years, while some of the studies reported median (IQR) values ranging from 25 (21–31) to 33.3 (29–37) years .Out of 21 studies, six reported that COVID-19 infection during pregnancy was not associated with adverse perinatal outcome ,30,31,32p < 0.05; I2 = 72 %). Based on 16 studies  [p < 0.05; I2 = 25 %). Out of 21 included studies, 20 studies  [p < 0.05; I2 = 95%). Only two studies provided data on termination of pregnancy [p = 0.37; I2 = 68 %). Pooled odds of 1358 COVID-19 infected pregnancies and 4045 non-infected pregnancies [p = 0.25; I2 = 65%)  ,32,33,34nancies) ,32,33,34regnancy ,22, but regnancy . Four stregnancy ,18,27,34gnancies ,31,32,332 = 65%) A.p < 0.05; I2 = 26%). Four studies [p = 0.36; I2 = 68%)  B.p < 0.05; I2 = 90%); pooled low birth weight [p < 0.05; I2 = 0%); pooled fifth minute APGAR score of less than 7 [p < 0.05; I2 = 0%) and pooled admissions to NICU [p < 0.05; I2 = 89%) were observed to be significantly higher. Nine studies [p = 0.05; I2 = 17%). Of the included studies, neonatal deaths were assessed in seven studies [p = 0.31; I2 = 0%)  C.Fifteen studies ,30,33,34p = 0.06; I2 = 65%). Gestational diabetes [p = 0.09; I2 = 83%) was also high among COVID-19 infected women compared to COVID-19 non-infected; however, the difference still was not significant. Pooled odds of 10,461 COVID-19 infected pregnancies and 496,246 COVID-19 non-infected in 17 studies revealed no statistically significant difference in chronic hypertension [p = 0.19; I2 = 38%) between COVID-19 infected and non-infected pregnant women. Data on asthma were reported by 14 studies, but only 12 studies [p = 0.64; I2 = 75%). Similarly, no statistically significant differences between COVID-19 infected and non-infected pregnancies were observed with relevance to anemia [p = 0.77; I2 = 0%), cardiac diseases [p = 0.98; I2 = 0%), chronic kidney disease [p = 0.45; I2 = 0%), chronic lung diseases other than asthma [p = 0.10; I2 = 0%), hypothyroidism [p = 0.85; I2 = 44%), immunosuppression [p = 0.80; I2 = 37%) and thrombophilia [p = 0.57; I2 = 0%)  .We conducted this systematic review to pool the available evidence of adverse perinatal outcomes caused by COVID-19 infection in pregnancy. We retrieved a total of 21 observational studies that assessed the adverse perinatal outcomes in pregnant women with COVID-19 infection published from December 2019 to June 2021. Overall findings of our study were, (1) the reported incidence rates of COVID-19 infection among pregnant women ranged from 1.3% to 27%, disregarding the fact that the results were based on single-center studies to multinational studies; (2) with regards to the adverse maternal outcomes, we found that there was a statistically significant increase in maternal deaths, preeclampsia, and cesarean deliveries, while miscarriages/abortions, PROMs/PPROMs, and operative vaginal births were non-significant in COVID-19 infected pregnant women compared to non-infected; (3) with regards to the adverse fetal outcomes, fetal distress was found to be statistically significant, while intrauterine death was non-significant in COVID-19 infected pregnancies; and (4) with regards to the adverse neonatal outcomes, all reported fetal outcomes except neonatal death, including preterm birth, low birth weight, stillbirth, fifth minute Apgar score < 7, and admissions to NICU showed significant differences in births to COVID-19 infected women compared to non-infected.The current study findings were consistent with previously published systematic reviews relevant to maternal death , preeclaHealthcare providers should be aware that women infected with COVID-19 have an elevated risk of disease severity, including maternal mortality. Pregnant women should be advised of the disease’s increased severity and encouraged to take precautions to avoid infection. Primary healthcare providers will need to balance the necessity for routine multidisciplinary prenatal care and the management of women suspected of having COVID-19 infection, preferably via virtual antenatal clinics. Pregnant women who become infected with COVID-19 before reaching term may require management in a tertiary healthcare facility equipped with cesarean section and NICU facilities to manage preterm infants, infants with low Apgar scores, and infants with fetal distress.This systematic review has several strengths. First, the study followed a sound methodology and was able to quantify the findings using meta-analyses. Second, a comprehensive search strategy was used to minimize the risk of missing relevant studies. Third, the screening was independently assessed by pairs of reviewers, and discrepancies solved by consensus. Fourth, excluding the publication types such as case studies, case reports, and case series left studies with a quality study design included in the final analysis. Finally, the present systematic review adhered to a rigorous quality appraisal. An important amount of evidence was summarized and critically appraised in addition to the highlighted evidence gaps.Our systematic review also has limitations. Firstly, the method of diagnosis of COVID-19 in pregnancy was different from study to study. Secondly, without data on disease severity, perinatal outcomes based on disease severity could not be determined. Thirdly, many studies represented developed countries with only meager contributions from low-resource countries. However, the findings of this systematic review have implications for low and middle-income countries with limited resources, where the negative impacts are prominent due to region-specific management strategies and resources. Finally, asymmetry of the funnel plots was observed for the assessed variables, and the presence of publication bias was suggested. This asymmetry may be also due to some other factors such as poor methodological design, reporting bias, chance or study heterogeneity. Despite all limitations, we undertook a comprehensive literature review and meta-analysis with the most updated findings relevant to adverse perinatal outcomes in COVID-19 infected pregnant women.Several adverse maternal, fetal, and neonatal effects were significantly higher in COVID-19 infected pregnant women than non-infected. These included maternal death, preeclampsia, cesarean section delivery, fetal distress, preterm birth, low birth weight, stillbirth, low Apgar score at the fifth minute, and admission to NICU. The comorbidity conditions had no added risk of being infected with COVID-19 infection during pregnancy. Therefore, a COVID-19 infected pregnant woman should be treated with special precautions to avoid and minimize the identified adverse events during perinatal care. Further studies are recommended to collect more robust data relevant to the adverse perinatal outcomes that will enable effective clinical decision-making in maternal and child health care.


Objectives: Sestrin2 is a stress-inducible protein and play an important role in adapting stress states of cells. This article reviewed the role of Sestrin2 in hypoxia and hypoxia-related diseases to provide new perspectives for future research and new therapeutic targets for hypoxia-related diseases.Methods: A review was conducted through an electronic search of PubMed and Medline databases. Keywords included Sestrin2, ROS, hypoxia, and hypoxia-related disease. Articles from 2008 to 2021 were mostly included and older ones were not excluded.Results: Sestrin2 is upregulated under various stress conditions, especially hypoxia. Under hypoxic condition, Sestrin2 plays a protective role by reducing the generation of ROS through various pathways, such as adenosine monophosphatea-ctivated protein kinase (AMPK) / mammalian target of rapamycin (mTOR) pathway and nuclear factor-E2-related factor2 (Nrf2) pathway. In addition, Sestrin2 is involved in various hypoxia-related diseases, such as cerebral hypoxic disease, myocardial hypoxic disease, hypoxia-related respiratory disease, and diabetes.Discussion: Sestrin2 is involved in various hypoxia-related diseases and maybe a therapeutic target. Furthermore, most studies focus on cerebral and myocardial ischemia reperfusion. More researches on hypoxia-related respiratory diseases, kidney injury, and diabetes are needed in future. In maCurrently, increasing studies focus on the relationship between hypoxia and Sestrin2 now that the hypoxia is an important inducer of stress ,7. Diver2.Recently, studies suggest that the Sestrin2 is regulated by several factors, such as P53, hypoxia inducible factor-1α (HIF-1α) and nuclear factor-erythroid 2-related factor2 (Nrf2), to exert a cells protective effect through promoting autophagy, inhibiting oxidative stress and ER stress . In thisIt has been widely reported that hypoxia-related diseases are related to Sestrin2 ,26. In hFurthermore, Sestrin2 promotes the activation of Nrf2 and degradation of Keap1 to exert antioxidant roles ,23. It w3.3.1.The neuronal cells are specifically assailable to hypoxia because of the unique structure and function, such as abundant presence of polyunsaturated fatty acid, high metabolism and dependence on oxygen . ConsequIncreasing studies suggest that Sestrin2 has important antioxidant effects in cerebral ischemia/reperfution (I/R) injury. The activation of Sestrin2 not only activates the AMPK pathway to inhibit mTORC1 and NOX4 to decrease the ROS accumulation ,32,39, bIn addition, many studies illustrate that Sestrin2 protect neuronal cells by promoting angiogenesis. Wang et al.  have dem3.2.2+ overload of cardiomyocytes or mitochondria, resulting in cell death [Myocardial hypoxia is considered to be a characteristic of the failing heart . Continull death ,49. Addill death .Present studies suggest that Sestrin2 plays an important role in cardioprotection against I/R injury, serving as an LKB1-AMPK scaffold to initiate AMPK activation during ischemic insults . SestrinMoreover, hypoxia causes the filtration of inflammatory factors which leads to dysfunction of the heart because of aseptic inflammation of cardiomyocytes . It is r3.3.Some respiratory diseases are usually accompanied by airway inflammation and airway obstruction, which lead to physiological dysfunction of the lung, and then make the body in a state of hypoxia ,60. TherAt present, increasing studies have found that Sestrin2 may exert a protective effect on hypoxia-related respiratory diseases. On the one hand, Sestrin2 maintains the integrity of airway epithelial cells . On the The role of Sestrin2 in COPD is highly complex. The inactivation of Sestrin2 increases the expression of PDGFR-β by inhibiting Nrf2/Keap1 pathway in a TGF-β-dependent manner to improve COPD by inhibiting oxidative stress or maintaining the integrity of airway epithelial cells ,62,63. SIn addition, the level of Sestrin2 is significantly higher in plasma and urine of OSA patients, and is positively correlated with AHI, even is helpful to the diagnosis of OSA ,65,66. MIt is reported that the level of Sestrin2 is significantly higher in asthma patientsand is independently correlated with the FEV1% predicted . It sugg3.4.Acute kidney injury (AKI) is a clinical syndrome with high incidence rate and poor prognosis ,69. It iAccumulating evidence suggests that hypoxia is an important pathogenic factor for diabetes or insulin resistance . Sestrin4.Sestrin2, as highly conserved stress-inducible protein, is involved in various hypoxia-related diseases and plays a protective role mainly through reducing production of ROS. The related pathways mainly include AMPK-mTOR, Keap1-Nrf2, Nrf2-HO-1 and other pathways. Therefore, Sestrin2 appears to be a therapeutic target for hypoxic-related diseases. Currently, most studies focus on cerebral and myocardial ischemia reperfusion. More researches on hypoxia-related respiratory diseases, kidney injury and diabetes are needed in future.


Epilepsy is one of the most common brain disorders worldwide. It is usually hard to be identified properly, and a third of patients are drug-resistant. Genes related to the progression and prognosis of epilepsy are particularly needed to be identified.In our study, we downloaded the Gene Expression Omnibus (GEO) microarray expression profiling dataset GSE143272. Differentially expressed genes (DEGs) with a fold change (FC) >1.2 and a P-value <0.05 were identified by GEO2R and grouped in male, female and overlapping DEGs. Functional enrichment analysis and Protein-Protein Interaction (PPI) network analysis were performed.In total, 183 DEGs overlapped (77 ups and 106 downs), 302 DEGs (185 ups and 117 downs) in the male dataset, and 750 DEGs (464 ups and 286 downs) in the female dataset were obtained from the GSE143272 dataset. These DEGs were markedly enriched under various Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) terms. 16 following hub genes were identified based on PPI network analysis: ADCY7, C3AR1, DEGS1, CXCL1 in male-specific DEGs, TOLLIP, ORM1, ELANE, QPCT in female-specific DEGs and FCAR, CD3G, CLEC12A, MOSPD2, CD3D, ALDH3B1, GPR97, PLAUR in overlapping DEGs.This discovery-driven study may be useful to provide a novel insight into the diagnosis and treatment of epilepsy. However, more experiments are needed in the future to study the functional roles of these genes in epilepsy. Epilepsy, one of the most common brain conditions including both genetic and acquired disorders, affects at least 46 million people worldwide . As a coDespite a number of genes and signaling pathways in the development and progression of epilepsy have been widely studied, the mechanisms underlying epilepsy are still being unraveled. Currently, the high-throughput sequencing analysis of gene expression, coupled with bioinformatics tools, becomes promising for investigating the novel genes in the initiation and evolution of diseases. In the study, we researched the human peripheral blood sample microarray dataset GSE143272 from the GEO to identify the DEGs between epilepsy patients and normal individuals by applying the bioinformatic method. Our results may provide potential biomarker candidates for clinical diagnosis and therapy of epilepsy.https://www.ncbi.nlm.nih.gov/geo/). The dataset was performed on GPL10558 Illumina HumanHT-12 V4.0 expression beadchip platform. The array data for GSE143272 contained 142 samples, including 34 newly diagnosed, drug-free patients with epilepsy, 57 followed-up patients receiving antiepileptic drug monotherapy, and 50 healthy subjects. We selected the drug-free epilepsy patients and healthy control subjects, consisting of 21 male epilepsy patients, 13 female epilepsy patients, 26 male controls, and 25 female controls. All data were downloaded from the GEO freely. No ethics approval and patients’ informed consent were needed for this present study.We downloaded the microarray expression profiling dataset GSE143272 as peripheral blood expression profiles of patients with epilepsy, deposited by Rawat C et al., from the GEO  to reveal the functions of DEGs, by conducting the analysis including GO annotation and KEGG pathway enrichment analyses. The GO analysis consisted of three divisions: biological process (BP), cellular component (CC), and molecular function (MF). P <0.05 and an enriched gene count ≥5 were selected as the criteria for statistical significance.We used the online database webgestalt (https://string-db.org/) to perform the PPI network analysis of evaluating the protein-protein interactions between the screened DEGs. The STRING, a PPI database, collects and assesses evidence from many sources, such as scientific literature, to integrate all known and predicted associations between proteins, including physical interactions and functional associations [We applied the online database STRING  DEGs. Besides, Sex-specific DEGs were screened, within which 302 (185 upregulated and 117 downregulated) DEGs were obtained in the male dataset, while 750 (464 upregulated and 286 downregulated) DEGs were obtained in the female dataset. The volcano plot and Venn diagram for the DEGs are presented in We carried out the functional enrichment analysis for the male-specific DEGs, female-specific DEGs, and overlapping DEGs. The upregulated and downregulated DEGs were analyzed respectively. Top enriched GO terms were shown in Figs In addition, the top KEGG pathways were presented in The STRING database was applied to determine the PPI networks with interaction score > 0.9 among the male-specific, female-specific, and overlapping DEGs, which were constructed via Cytoscape software, respectively Figs –7.Cytohubba plugin of Cytoscape was used to rank the top 10 nodes in the above PPI networks according to 3 topological analysis methods, including MCC, MNC, and Degree. The overlapping hub genes according to the three methods were ADCY7, C3AR1, DEGS1, CXCL1 in male-specific DEGs, TOLLIP, ORM1, ELANE, QPCT in female-specific DEGs, and FCAR, CD3G, CLEC12A, MOSPD2, CD3D, ALDH3B1, GPR97, PLAUR in overlapping DEGs .In this study, we performed bioinformatics analysis to search for the potential key genes associated with epilepsy. Male and female epilepsy were compared to healthy controls respectively on the hypothesis that epilepsy in different genders had different mechanisms. The results showed that 302 male-specific DEGs, 750 female-specific DEGs, and 183 overlapping DEGs were successfully identified. Those DEGs were put into multi-step bioinformatic functional annotations, including GO, KEGG, and PPI analysis.We observed that male, female and overlapping DEGs took part in similar and overlapping biological processes significantly. Most of the biological processes were involved in inflammation and immune system defense response, for example, immune response, leukocyte mediated immunity, T cell activation, etc. In KEGG analysis, sphingolipid signaling pathway was showed upregulated in male patients but downregulated in female patients. It is well known that sphingolipids, such as ceramide, sphingosine, sphingosine-1-phosphate, sphingomyelin, and gangliosides, play an important role in the regulation of steroidogenesis . The sexBased on the PPI networks, 16 hub genes were identified, which were ADCY7, C3AR1, DEGS1, CXCL1 in male-specific DEGs, TOLLIP, ORM1, ELANE, QPCT in female-specific DEGs and FCAR, CD3G, CLEC12A, MOSPD2, CD3D, ALDH3B1, GPR97, PLAUR in overlapping DEGs, respectively.ADCY7 encodes a membrane-bound adenylate cyclase (AC), which is one of the most ubiquitous signal transduction molecules that catalyzes the formation of cyclic adenosine monophosphate (AMP) from adenosine triphosphate (ATP) , 21. It C3AR1 is a Protein Coding gene. The protein C3a Receptor 1 encoded by this gene is an orphan G protein-coupled receptor for C3a, which is a proinflammatory mediator released during activation of the complement system . The comDEGS1 encodes Delta 4-Desaturase, Sphingolipid 1, a member of the membrane fatty acid desaturase family which is responsible for inserting double bonds into specific positions in fatty acids. The related pathways of DEGS1 are sphingolipid signaling pathway and sphingolipid metabolism , 42. In CXCL1 gene encodes a member of the CXC subfamily of chemokines. The CXCL1 protein plays a pivotal role by recruiting and activating neutrophils in inflammation when signaling through the CXCR2 receptor . It is bTOLLIP encodes the Toll-interacting protein (Tollip), which is present in a complex with the interleukin-1 receptor associated kinase (IRAK). With the activation of IL-1β, the Tollip–IRAK complex is recruited and disrupted . The oveORM1, accounting for 75% of plasma ORM, is an inflammatory factor with multiple activities . Plasma ELANE encodes neutrophil elastase (NE), which belongs to the family of serine proteases. It has been reported that the mutations in the gene usually cause Cyclic neutropenia (CyN) and severe congenital neutropenia (SCN) , 71. As The QPCT gene encodes glutaminyl cyclase (QC). QC is an enzyme responsible for catalyzing the posttranslational modification of N-terminal glutamate to pyroglutamate in many neuroendocrine peptides, which renders the protein more susceptible to neurotoxic . AmyloidFCAR encodes FcαRI or CD89, which is expressed on cells of the myeloid lineage  and is aCD3G and CD3D encode CD3γ and CD3δ, respectively. Both CD3γ and CD3δ are part of the T-cell receptor/CD3 complex (TCR/CD3 complex) , which iCLEC12A is a C-type lectin receptor (CLR) and a Src homology region 2 domain-containing phosphatase 1 and 2 (SHP-1 and -2)-associated receptor, highly expressed on human dendritic cells (DCs). CLEC12A contains a single immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic tail. In response to chemokine CCL2, ITIM can associate with SHP-1 and SHP-2, involved in inhibitory signaling as a key molecule to deliver immature DCs to the CNS across the BBB . It has MOSPD2 (motile sperm domain-containing protein 2) is the surface protein predominantly expressed on cytoplasmic membrane of human monocytes. MOSPD2 is also found in neutrophils, but not in lymphocytes. It is revealed that MOSPD2 is critical in regulating the inflammatory monocyte and neutrophil migration without activating ligands . The monALDH3B1 encodes the protein that belongs to the ALDH3 protein family (Aldehyde Dehydrogenase Family 3) –111. It GPR97 belongs to the G protein-coupled receptors (GPCRs), the largest receptor superfamily broadly involved in the regulation of biological processes and various diseases, including CNS disorders, such as anxiety, depression, schizophrenia, epilepsy, Alzheimer’s disease, and Parkinson’s disease , 129. GPPLAUR encodes urokinase-type plasminogen activator receptor (uPAR) , which iSummarily, using the profile dataset and bioinformatics analysis, 16 epilepsy-associated hub genes were identified . HoweverIn conclusion, by using the integrated bioinformatics analysis for gene expression profiles in epilepsy, we identified 16 hub genes, including sex-specific genes. These hub genes were correlated with the pathogenesis and prognosis of epilepsy. This study may contribute to further insight into epilepsy, by digging out the potential diagnostic and prognostic biomarkers, as well as therapeutic targets. Nevertheless, in the future, more research (in vivo and in vitro experiments) should be carried out to validate the functional roles of these genes in epilepsy.S1 Data(XLSX)Click here for additional data file.


Pancreatic cancer (PC) is a lethal malignancy cancer, and its mortality rates have been increasing worldwide. Diagnosis of this cancer is complicated, as it does not often present symptoms, and most patients present an irremediable tumor having a 5-year survival rate after diagnosis. Regarding treatment, many concerns have also been raised, as most tumors are found at advanced stages. At present, anticancer compounds-rich foods have been utilized to control PC. Among such bioactive molecules, flavonoid compounds have shown excellent anticancer abilities, such as quercetin, which has been used as an adjunctive or alternative drug to PC treatment by inhibitory or stimulatory biological mechanisms including autophagy, apoptosis, cell growth reduction or inhibition, EMT, oxidative stress, and enhancing sensitivity to chemotherapy agents. The recognition that this natural product has beneficial effects on cancer treatment has boosted the researchers' interest towards more extensive studies to use herbal medicine for anticancer purposes. In addition, due to the expensive cost and high rate of side effects of anticancer drugs, attempts have been made to use quercetin but also other flavonoids for preventing and treating PC. Based on related studies, it has been found that the quercetin compound has significant effect on cancerous cell lines as well as animal models. Therefore, it can be used as a supplementary drug to treat a variety of cancers, particularly pancreatic cancer. This review is aimed at discussing the therapeutic effects of quercetin by targeting the molecular signaling pathway and identifying antigrowth, cell proliferation, antioxidative stress, EMT, induction of apoptotic, and autophagic features. Pancreatic cancer (PC) is an increasingly common cancer of the gastrointestinal tract (GIT), with survival rates less than 5% at 5 years after diagnosis, and about 50% of all patients die over 6 months of diagnosis. According to estimations in the United States, PC will become the second most common cause of cancer death in the next twenty to thirty years. However, patients' prognosis with localized and respectable tumors remains poor with only 20% survival rate after surgery . In addi15H10O7) is called by IUPAC  as follows: 3,3,4,5,7-pentahydroxyflavone and 2--3,5,7-trihydroxychromen-4-one [PC is characterized as a tumor of the exocrine pancreas and ductal adenocarcinoma; however, a minor subset of patients also represents neuroendocrine tumors. Indeed, pancreatic intraepithelial neoplasia or precursor lesions are the operative factors in the acquisition of genetic shifts which trigger discernible pancreatic ductal adenocarcinoma (PDA) . Unfortuen-4-one . It has en-4-one . Specifien-4-one , 14. Noten-4-one –19. Addien-4-one , commercCurrently with an average 5-year survival rate, PC is estimated to be the second cause of cancer-related deaths by 2030 in the United States –23. The An earlier diagnosis would be very helpful in the successful treatment of this malignancy, despite the scarce presence of symptoms among individuals. Regarding treatment, surgery, chemotherapy, and radiotherapy are the most common therapeutic strategies applied for PC treatment. Actually, the standard course of treatment is surgery following adjuvant therapy; however, the recurrence of 70-80% of resected tumors ultimately occurs. Patients who are eligible for surgical resection comprise merely almost 10-15% of all patients with advanced PC. With the majority of patients being diagnosed at later stages, chemotherapy remains as only treatment option for PC. 5-Flourouracil (5-FU) and gemcitabine (GMC), alone or in combination with radiation, are the standard chemotherapy regimen for PC's treatment, even though the response rate is usually below 31%. GMC has some advantages over 5-FU, such as the ability to relieve most disease's symptoms and having a modest survival advantage; however, it could not extend the average survival rate much beyond 6 months, like other chemotherapeutics , 29. HenκB, and cytochrome C signaling pathways [κB activation by the suppression of TGF-β and inhibitor of IκB kinase (IKK) activation in HNSCC cells [Various observational and prospective studies have revealed an indirect association between fruit and vegetable consumption with the occurrence of some cancers and the great potential of natural compounds to change the natural history of carcinogenesis –32. Planpathways , 53. OneCC cells . In addiCC cells . It can CC cells .Among the plethora of biologically active phytochemicals with anticancer potential, they are chemically categorized into phenolics, carotenoids, phytosterols, organosulfur compounds, and nitrogen-containing compounds , 57. Phe3, the position in quercetin structure, also adds to its free radical scavenging ability [μM once being absorbed in the form of glucosides. However, its peak plasma concentration is less than 0.33 μM when absorbed in the unconjugated form, showing less efficient absorption [Belonging to the class of polyphenolic flavonoid compounds and the subclass of flavonols, quercetin is ubiquitous in daily foods, including various plants, vegetables, nuts, seeds, fruits, tea, and red wine , 65. How ability , 71. The ability . It has  ability . The bio ability . Quercet ability . In plan ability . These q ability –75. The sorption .+ and CD133− pancreatic cancer cells. CD133+ cells were obtained from the PANC-1 cells by the MiniMACS system. CD133+ and CD133− PANC-1 cells were treated with different concentrations of resveratrol and quercetin. Immunocytochemistry tests using antibodies such as TNF-α, ACTA-2, N-cadherin, IL-1β, and vimentin were applied for assessing the anticancer and antimetastatic properties of resveratrol and quercetin. Results revealed that the immunostaining intensity of CD133+ cells was stronger than that of CD133− cells. ACTA-2, N-cadherin, and IL-1β immunoreactivities were significantly decreased, whereas vimentin and TNF-α immunoreactivities increased in quercetin treated CD133+ cells. In addition, quercetin was more effective than resveratrol in inhibiting metastasis. Guo et al. [β1 level, which inhibited the PCC migration and invasion. Quercetin treatment reduced the PDA growth and metastasis in nude mouse models by decreasing SHH activity. Additionally, SHH activated TGF-β1/Smad2/3 signaling and stimulated EMT by inducing the expression of Snail1and Zeb2 that instigated a partial reversal of quercetin-mediated inhibition of PCC migration and invasion.Quercetin has numerous benefits on human health, including anticancer, antioxidant, antidiabetic, antiulcer, anti-inflammatory, antiviral, antiallergic, antihypertension and anti-infection, cardioprotective, gastroprotective, and immune-modulatory effects , 77. Wito et al.  have stuGMCR cells). Moreover, quercetin reduced RAGE expression and facilitated the apoptosis, autophagy, and chemosensitivity to GMC in MIA Paca-2 GMCR cells, which suggests that further cytotoxicity has been achieved by the addition of quercetin in treatment with GMC. Yu et al. [According to Pang et al. , quercetu et al.  showed tu et al.  have expin vitro and xenograft models of PC. According to the results, after treatment with BET inhibitors and quercetin the proliferation and sphere-forming ability of cancer cells was reduced, and apoptosis stimulated. In addition, quercetin diminished the nuclear protein hnRNPA1 which control mRNA translation and export of antiapoptotic proteins, in vivo and increased the BET inhibitors effects at suppressing cells proliferation and tumor growth. In another study, Nwaeburu et al. [In vivo xenotransplantation of PDA cell and following IV injection of let-7c provoked a noteworthy reduction of tumor mass in the fertilized chick egg model. Immunohistochemistry analysis demonstrated that let-7c upregulated the Numbl and reserved Notch and progression markers. The findings illustrate that Que-induced let-7c declines cancer cell divisions and tumor growth.Inhibition of PC cell proliferation could signify a distinct mechanism of anticancer effects of quercetin . In thisu et al.  explored2O2 [Redox homeostasis is very important for cell function and ROS have an essential role in cell signaling. However, the disturbance in the antioxidant system could lead to excessive intracellular ROS levels, such as hydroxy free radicals and H2O2 . The exc2O2 –100. Theπ orbitals allowing it to donate hydrogen or electrons, and thus scavenge superoxide anion (•O2−) and H2O2. Quercetin is regarded as outstanding free-radical scavenging antioxidant [2O2 by reaction with •O2−, while also decreases H2O2 levels in the presence of peroxidases and keeps cells safe against H2O2 damages [With the numerous OH groups and conjugated ioxidant . Quercet damages . The sem damages . QQ is h damages , 102. QQ damages , 104; th damages . Consequ damages , 106. Th damages . Besides damages , 109. Mo damages , 111.β/Smad signaling pathways, involved in promoting EMT by the induction of Zeb2 and Snail1 expressions [α-SMA) gene expressions, and upregulated Cdh1 (encoding E-cadherin) gene expression in PANC-1 and Patu8988 cells; upon quercetin treatment, the protein levels of type I collagen, N-cadherin, α-SMA and vimentin were reduced,; however, the protein level of E-cadherin was increased in cells [β1 expression and that of EMT-TFs (EMT-inducing transcription factors) Snail1 and Zeb2 [β1/Smad2/3 signaling pathway suppressing E-cadherin expression [β1 and forming heteromeric complexes with Smad4, Smad2 and Smad3 translocate to the nucleus and induce EMT-TFs' expression [A physiological process, epithelial-to-mesenchymal transition (EMT), has an key function in mammalian embryonic development and cell and tissue balance; however, it has also an important role in tumorigenesis and tumor progression . During ressions . Quercetin cells . Quercetand Zeb2 . EMT-TFspression , 119. Fupression . It has pression . It has pression . Quercetpression , suggestpression .GEMR cell via the PI3K/AKT/mTOR axis [By the improved effectiveness in combination with other dietary agents, quercetin has been investigated as a promising adjuvant to increase the effectiveness of numerous chemotherapeutics , 123. LaTOR axis . As RAGETOR axis , proposiTOR axis , and decTOR axis . It has TOR axis , 126. OnTOR axis , 126. BoTOR axis  indicateTOR axis . They alTOR axis .Food consumption combined with therapeutic agents has been considered a key for the successful treatment of several diseases, including cancer. Conventional therapies like natural components besides other therapeutic methods due to their lower cost and side effects have been increasingly considered by researchers. Specifically, quercetin exerts an anticancer effect against PC cancer cells by mediating apoptosis, but recent studies have also indicated that quercetin affects various signal transduction pathways to reduce cancer progression. Quercetin suppresses the expression of N-cadherin, MMP-9, STAT-3 signaling pathways and potentially inhibits EMT, invasion, and metastasis. Quercetin enhances gemcitabine chemosensitivity in pancreatic cancer cells through the inhibitory effect on RAGE expression. Meanwhile, it has wide accessibility, efficacy and low toxicity comparing with other studied compounds, make it an appealing agent in cancer treatment. More recently, quercetin has been introduced and applied as a promising drug in the treatment of various cancers alone or in combination with other chemotherapeutic agents. Future well-designed clinical studies are needed to help the scientists to evaluate the safety and potential of quercetin against PC.


Aberrant glycosylation is a key feature of malignant transformation. Hypersialylation, the enhanced expression of sialic acid-terminated glycoconjugates on the cell surface, has been linked to immune evasion and metastatic spread, eventually by interaction with sialoglycan-binding lectins, including Siglecs and selectins. The biosynthesis of tumor-associated sialoglycans involves sialyltransferases, which are differentially expressed in cancer cells. In this review article, we provide an overview of the twenty human sialyltransferases and their roles in cancer biology and immunity. A better understanding of the individual contribution of select sialyltransferases to the tumor sialome may lead to more personalized strategies for the treatment of cancer. Cancer remains one of the leading cause of death worldwide . During Commonly found glycosylation changes in cancer cells include hypersialylation, incomplete synthesis, truncation of O- and N-glycans, altered branching, and even xenoglycosylation , 13. HypIn humans, twenty different sialyltransferases (SiaTs) are involved in the biosynthesis of glycans and each exhibits distinct characteristics and preferences such as for substrates and glycosidic linkages. The expression levels of individual SiaTs varies significantly between different types of tumors , but alsSialic acids (neuraminic acids) are nine-carbon (C1-9) monosaccharides most commonly found at a terminal position on the outer end of glycoconjugates on many glycoproteins and glycolipids synthesized by living cells . Their pCMAH (Cytidine monophospho-N-acetylneuraminic acid hydroxylase) gene, which is responsible for the conversion of Neu5Ac into Neu5Gc  and N-glycolylneuraminic acid (Neu5Gc) monosaccharides  Figure, 27. As o Neu5Gc   by UDP-GlcNAc 2-epimerase/ManNAc-6 (GNE) in a two-step process (via the action of cytosine 5’-monophosphate N-acetylneuraminic acid synthetase (CMAS) to produce CMP-Neu5Ac  from sialylated glycoconjugates . There ain vitro with recombinant enzymes revealed that one linkage can be synthesized by multiple enzymes  as an activated sugar donor for the transfer of sialic acids to the terminal glycosyl group of glycoproteins and glycolipids as acceptor molecules . SiaTs c enzymes , 48. Sia enzymes . The L-m enzymes . A disul enzymes .SiaTs have been shown to be primarily restricted to medial- and trans-cisternae of the Golgi apparatus, with some being present in the trans-Golgi network , but somIncreased activity or expression of SiaTs leads to the hypersialylation of cell surfaces which is one of the most common glycosylation changes that occurs in tumors; it entails the enhanced expression of sialic acid-terminated glycoconjugates . Many stSix β-galactoside α2,3-sialyltransferases belong to the ST3Gal family in humans and these enzymes transfer sialic acid residue in an α2,3-linkage to terminal galactose  residues present on glycolipids or glycoproteins , 60. MemO-glycans such as in hematopoietic cells  antigen from the T antigen Galβ1-3GalNAc. While the T and ST antigens are found on normal ic cells , ST3Gal1ic cells –65, and ic cells . MUC1-STic cells , throughic cells . Recentlic cells . Overexpic cells –72, whicic cells . Moreoveic cells . ST3Gal1ic cells , paclitaic cells , and tamic cells . The exaIn vivo genetic experiments showed that ST3Gal2 is a key enzyme mediating α2,3 sialylation of gangliosides in the brain of mice, in particular of GD1a and GT1b, eventually with support of ST3Gal3  and sLeX, which are expressed in different types of cancer –87, and rognosis . ST3Gal3rognosis , 92.ST3GAL4 expression correlates with enhanced metastatic potential and poor prognosis in some types of cancer, including pancreatic and gastric cancer  . ST6Gal1sistance . Indeed,sistance , cisplatsistance , trastuzsistance , 141 or sistance , latter sistance .Several investigators observed that α2-6 sialylation by ST6Gal1 activity may protect cells from cell death, and eventually block homeostatic epithelial cell apoptosis in cancer . ST6Gal1Recently, using gene engineered HEK293 cells, ST6Gal1 was found to be partially responsible for the generation of ligands for the immunoregulatory receptor Siglec-7 , indicatST6GAL2 in breast cancer cells resulted in reduced xenograft tumor growth in vivo  and metastasis pathways proteins . Similarly, silencing of ST6GAL2 in FTC reduced tumor growth in an in vivo model  residues found on ST6GalNAc1 catalyzes the generation of sialyl-Tn (sTn) antigen from Tn antigen . sTn is ST6GALNAC1 via hyper-methylation and loss of heterozygosity (LOH) was observed in esophageal carcinoma in tylosis, an inherited epithelial disorder  adhesion, migration and invasion in vitro , 166, an were shown to be key in conferring tumor cell glycosylation changes that contribute to metastatic activity in a primary lung cancer model, eventually by preserving presentation of the T-antigen and adherence to galectin 3 , and also generates the disialyl-lactotetraosyl-ceramide GD1α from sialyl-lactotetraosyl-ceramide GM1b (gangliosides) yet to a lesser degree than ST6GalNAc3 , 182. Uplectin 3 . In anot in vivo .ST6GALNAC5 determine susceptibility for colorectal brain metastasis and overall survival  silenced cells exhibited reduced blood brain barrier (BBB) transmigration activity , as it catalyzes the transfer of a sialic acid residue onto GM3 to give raise to the b-series ganglioside GD3, which can eventually be further processed for the biosynthesis of other b-/c-series gangliosides . GD3S exIA1 gene . As oppoIA1 gene . HoweverIA1 gene . FurtherIA1 gene . ST8Sia1IA1 gene , 209. InIA1 gene . The impIA1 gene . ST8Sia1IA1 gene , 210, whIA1 gene , 82, andIA1 gene .in vivo model, ST8Sia2-transfected glioma cells with high expression of polySia exhibited increased tumor invasion within the brain of recipient mice  is involved in the synthesis of linear polymers of sialic acid, so-called polysialic acid (polySia) chains . Polysiaent mice . Overexpin vitro . Cytidinin vitro . ST8Sia2in vitro .ST8SIA3 knockdown experiments in vitro  and select glycoproteins, including striatal glycoproteins , 220. STin vitro . Moreovein vitro, and decreased tumor growth in vivo. High levels of ST8Sia4 expression was observed in chemoresistant leukemic cells , synthesizes slightly longer polySia chains compared to ST8Sia2, eventually conferring different molecular properties . Both poic cells –228, whiic cells , 227. Ho in vivo . Specifi in vivo , 230, anST8SIA5 transcript was also observed in a murine model of colitis-associated cancer , are a family of I-type lectins that belong to the immunoglobulin superfamily. Siglecs are cell-surface receptors predominantly expressed on leukocytes in a cell-specific and differentiation-dependent manner . On the in vitro, and in a Siglec humanized in vivo model  lectins comprising E-selectin, L-selectin, and P-selectin, named after their expression on endothelial cells, leukocytes and platelets. In contrast to L-selectin that is constitutively expressed on leukocytes and E-selectin in postcapillary venules of the skin and bone marrow , howeversferases . As discIn vivo studies using selectin knockout or selectin ligand deficient mice highlighted the importance of selectins in metastasis , can vary between different types of tumors, between primary tumor and metastatic lesion, and even between patients . Furtheriews see –259], coiews see , and morAll authors wrote and approved the manuscript.The laboratory of SV is supported by grants from the Swiss National Science Foundation (310030_184757), the Swiss Cancer League/Swiss Cancer Research (KFS-4958-02-2020), and the Bern Center for Precision Medicine (BCPM).SVG received remuneration for serving on the scientific advisory board of Palleon Pharmaceuticals.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Introduction: Treatment options for biliary tract cancer (BTC) are very limited. It is necessary to investigate actionable genes and candidate drugs using a sophisticated knowledgebase (KB) and characterize BTCs immunologically for evaluating the actionability of molecular and immune therapies.®). Predictive biomarkers of immune checkpoint inhibitors were analyzed using DNA and RNA sequencing data.Materials and Methods: The genomic and transcriptome data of 219 patients with BTC who underwent surgery were analyzed. Actionable mutations and candidate drugs were annotated using the largest available KB of the Asian population (CancerSCANPTEN (7.3%), CDKN2A (6.8%), KRAS (6.4%). BRCA2, CDKN2A, and FGFR2 mutations were most frequently identified in case of intrahepatic cholangiocarcinoma. PTEN and CDKN2A mutations were associated with significantly shorter overall survival. PD-L1 and PD-1 expression was significantly higher in case of extrahepatic cholangiocarcinoma and T-cell-high expression. In total, 49.7% of cases were evaluated as having actionability for molecular therapy or immune checkpoint inhibitors.Results: Twenty-two actionable genes and 43 candidate drugs were annotated in 74 patients (33.8%). The most frequent actionable genes were Conclusions: Identifying actionable genes and candidate drugs using the KB contribute to the development of therapeutic drugs and personalized treatment for BTC. Biliary tract cancer (BTC) or cholangiocarcinoma, which originates from bile duct epithelial cells (cholangiocytes), is a rare tumor worldwide. However, it is prevalent in some areas with a high incidence of specific risk factors such as chronic inflammation of the biliary tract and gallbladder and hepatitis, as well as chemical exposure such as aflatoxin , 2. AccoTP53, KRAS, SMAD4, ARID1A, CDKN2A, IDH1, and ELF3 mutations ), followed by perihilar cholangiocarcinoma (PHC), distal cholangiocarcinoma (DCC), and gallbladder carcinoma (GBC), including cystic ductal carcinoma (CDC). The most common pathological staging was stage III (36.1% [n = 79]), and 35.2% (n = 77) had lymph node metastases.We accumulated the data of WGS, WES, targeted sequencing, and RNA-seq from BTCs  and ICCsWe annotated the actionable mutations using a KB containing the frequency data of approximately 15,000 East Asians as well as drug mapping information. Using this KB, we analyzed 369 genes for SNVs, indels, and CNVs using genomic DNA and the fusions of 627 genes using capture-based RNA-seq. The list of genes used in the analysis is listed in Supplementary Tables 2 and 3. WGS, WES, and targeted sequencing revealed 602 SNVs and 28 indels in protein-coding regions and splice sites, respectively, and 512 non-coding mutations. We annotated 41 SNVs and one indel using the KB for actionability (Supplementary Table 4). WGS revealed 392 CNVs and we annotated 33 CNVs; WES revealed 635 CNVs of which we annotated 46 CNVs (Supplementary Table 5). RNA-seq revealed 51 fusions and of which 15 fusions were annotated (Supplementary Table 6).PTEN (7.3%), followed by CDKN2A (6.8%), KRAS (6.4%), PIK3CA (4.1%), and MDM2 (4.1%). All variants of PTEN showed copy number loss (Supplementary Table 5). Only FGFR1/2 were found as the actionable gene caused by fusion.Aggregating these results, 22 actionable genes and 43 candidate drugs were annotated in 74 cases using the KB . In total, 33.8% of the BTC cases had at least one actionable mutation.Actionable genes were aggregated in the following pathways : MAPK paP = 0.001). BRCA2, CDKN2A, and FGFR2 mutations were most frequently identified in ICC . In contrast, 73.2% of ECCs were found to have no actionable mutations. Pathological lymph node metastasis and sex were examined, and no significant differences in actionable genes were identified (Supplementary Tables 8 and 9).Next, the relationship between actionable mutations and clinicopathological features in BTC was investigated. Based on the anatomical location of BTC in pathological diagnosis , PHC, GBn = 208) of the cases and for relapse-free survival (RFS) in 94% (n = 206) of the cases. Kaplan–Meier curves for OS and RFS were plotted for actionable genes with mutations in more than five cases and were analyzed using the log-rank test    and RFS tended to be shorter (P = 0.06). No other genetic alterations were found to shorten or prolong the OS and RFS.Prognostic data were available for overall survival (OS) in 95% (ctively)  and 2B. ctively)  and 2D. TMB was calculated and used as a predictive biomarker for ICI. Of the 147 patients for which WGS and WES were performed, six cases (4%) were classified as TMB-High . The TMBPD-L1 and PD-1 expression may be predictive biomarkers for ICIs. Here, we investigated the expression of predictive ICI biomarkers based on the RNA-seq results. The clinicopathological data of 115 cases with RNA-seq are shown in Supplementary Table 11. T-cell signature genes [PD-L1, and PD-1 expression levels were annotated on the heat map, the T-cell-high group tended to have more ECC and higher PD-L1 and PD-1 expression.In addition to TMB, T cell expression and re genes  were cluPD-L1 expression was associated with pembrolizumab response [PD-L1 and PD-1 expression and the location of BTC, sex, and T-cell expression were investigated . PD-1 expression was significantly higher in ECC, males, and T-cell-high expression .Based on the report that high response , the relstigated . PD-L1 ePDL-1 or PD-1 expression. Assuming T-cell-high expression as an indicator, 35 cases had the actionability for ICI. Four patients had actionability for both molecular therapy and ICI. In total, 47.9% (105/219 cases) of BTC cases had actionability for molecular therapy or ICIs.There were stronger associations between T cell hyper-expression and high PTEN and CDKN2A were most frequently detected among the annotated actionable genes, and their mutations affected OS and RFS. We also characterized the biomarkers of ICIs, TMB, T cell expression, PD-L1, and PD-1 in BTCs using RNA-seq to evaluate the immunological actionability for BTC.In this study, 22 actionable genes and 43 candidate drugs were identified in 219 BTCs using the Asian KB. ERBB2 and ramucirumab targeted at VEGFR have been observed to be effective for treatment [Reports that estimated actionable genes using surgical specimens of BTC showed frequencies of 25% and 38.9%, respectively, , 24, whireatment . Thus, dKRAS mutation representative of the MAPK pathway is identified in 11–27% of BTC [KRAS, has been studied and has shown encouraging anticancer activity in phase I trials in patients with KRAS p.G12C mutation-positive advanced solid tumors [In this study, actionable genes in the MAPK pathway were observed to be the most frequent. % of BTC –8. The M% of BTC  have beed tumors .PTEN and CDKN2A were the most frequently identified actionable genes in this study. Notably, PTEN and CDKN2A were associated with worse OS and RFS. The prognostic effect of these variants has been reported previously [PTEN, a tumor suppressor gene, is a negative regulator of the PI3K/mTOR pathway, and abnormal activation of the PI3K/mTOR pathway has been associated with the development of malignancies, including BTC [CDKN2A is also a tumor suppressor gene that encodes the CDK4/6 inhibitor p16ink4a [CDKN2A aberrations have been reported in 32% of ECCs and 28% of ICCs, making CDKN2A a new therapeutic target of interest [in vitro [In contrast, eviously , supportding BTC . GSK2636ding BTC  and AZD8ding BTC , the PI3ding BTC . CDKN2A p16ink4a . Loss ofinterest . The CDKin vitro .IDH1 mutations (ClarIDHy) [IDH1 mutations had ICC. In a clinical trial of FGFR2 mutations (FIGHT202) [FGFR2 fusion or rearrangements had ICC. These trials have demonstrated the clinical benefits of molecularly targeted drugs. In this study, IDH1 was observed only in ICC. FGFR2 was more frequently observed in ICC. Furthermore, the overall actionable genes were more frequently observed in ICC. The results of this study thus indicate that ICC may benefit more from molecular-targeted therapy.Clinical trials of new drugs for BTC tend to focus on ICC. In a clinical trial of larIDHy) , 90% of IGHT202) , 98% of PD-L1 and PD-1 expression. Fontugne et al. [PD-L1 expression and T cell infiltration in BTC, supporting the results of this study. Kriegsmann et al. showed that PD-L1 expression in BTC cells was comparable between ICC (5%), PHC (4%), and DCC (3%), whereas PD-L1 expression in stromal cells was the highest in DCC  [PD-L1 expression in ECC in this study may be because of the high PD-L1 expression in stromal cells. Pembrolizumab for head and neck squamous cell carcinoma has shown efficacy in patients with positive PD-L1 expression in cancer tissues, including stromal cells [This study also characterized the biomarkers of ICI and focused on e et al.  reportedal cells . Thus, pThis study had some limitations. First, the analysis was performed on surgical specimens and may not reflect actionable genes in advanced BTC. Second, the sequencing method was not uniform in this study. The TMB-High thresholds were set separately, but there were differences in the frequency of TMB-High between WGS/WES and targeted sequencing, which may have led to misclassification. A uniform sequencing method should be used to re-evaluate TMB. Finally, this cohort did not receive any of the candidate drugs annotated in this study; therefore, the usefulness of candidate drugs needs to be assessed.However, due to the rarity of BTC, large-scale clinical trials are challenging to establish. Recently, it was demonstrated that BTC cell lines and organoids have genomic alterations similar to those in primary tumors, indicating that they could be useful in developing and validating therapeutic targets for BTC . As showIn conclusion, we identified 22 actionable genes and 43 candidate drugs for BTC using a KB with research-level information on genomic alterations. We also characterized the biomarkers of ICIs using RNA-seq. Further validation of comprehensive candidate drugs using cell lines and organoids based on these data, may facilitate drug discovery for BTC.Two hundred and nineteen patients diagnosed with BTC were enrolled at Hokkaido University Gastroenterological Surgery II between 2003 and 2018. Two hundred and eighteen patients with BTC were enrolled in the International Cancer Genome Consortium (ICGC)  for all 107 cases that underwent WES. For WGS, DNA was extracted from the tumor and normal tissue, and 500–600-bp insert libraries were prepared following the manufacturer’s protocol. The exome-capture or WGS libraries were sequenced on HiSeq2000/2500 with paired reads of 100–125 bp. Targeted sequencing was performed on Illumina HiSeq2000/2500 after capturing using the SureSelect XT Custom kit (Agilent Technologies) following the manufacturer’s protocol. RNA-seq libraries were prepared using the TruSeq Stranded mRNA Sample Prep kit (Illumina) or the KAPA RNA HyperPrep Kit with RiboErase (Roche) following the manufacturer’s protocol. The Truseq Stranded mRNA Sample Prep Kit was used in 87 cases, and the KAPA RNA HyperPrep Kit with RiboErase was used in the other 28 cases. RNA-seq was performed on the HiSeq2500 platform.https://genomon.readthedocs.io/ja/v2.6.1/). Briefly, sequence reads were mapped to the human reference genome GRCh37 using BWA [http://broadinstitute.github.io/picard), and after comparing the results from cancerous and normal tissues, SNVs and indels were detected using Genomon2. Copy number variation (CNV) in WGS and WES was calculated using Sequenza R [https://github.com/Genomon-Project/fusionfusion).Single nucleotide variants (SNVs) and indels were detected using the in-house pipeline Genomon2  in particular. Using this database, tier information that maps drugs to tumor types can be obtained based on the ACMG guidelines. In addition, the KB can be used throughout cancer genome analysis, such as for predicting the actionability for variants of unknown significance (VUS) through variant frequency information.CancerSCAN®  is a plaBRAF, HRAS, KRAS, and NF1; receptor tyrosine kinase (RTK), ALK, EGFR, ERBB2, FGFR1, FGFR2, FGFR3, FLT3, and MET; phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling, PIK3CA, PTEN, and TSC1; cell cycle control, CDKN2A and CDK4; DNA damage repair, ATM, BRCA1, and BRCA2; p53 signaling pathway, MDM2; and metabolic pathway, IDH1.The actionable genes found to be altered in this study were classified in terms of the pathways involved based on the literature as follows , 48: mitTMB is defined as the number of nonsynonymous somatic mutations, coding mutations, base substitution mutations, and indel mutations per megabase (Mb) of the genome. When calculating the TMB, we divided the total number of mutations by 39 Mbp for WGS and WES and by 0.196 Mbp for targeted sequencing. There are still no clear criteria for the cut-off value for TMB-High. The cut-off value for TMB-High of pembrolizumab for solid tumors, approved by the Food and Drug Administration (FDA) in 2020, was 10 mutations/Mbp ; in the CD8A, CCL2, CCL3, CCL4, CXCL9, CXCL10, ICOS, GZMK, IRF1, HLA-DMA, HLA-DMB, HLA-DOA, and HLA-DOB), PD-L1 (CD274), and PD-1 (CD279) in 115 patients who underwent RNA-seq were determined. RNA-seq reads were mapped to the reference sequence using STAR [To predict the effect of ICIs, the expression levels of T cell signature genes  . Continuous variables are expressed as median, minimum, and maximum. Nominal variables are expressed as frequencies and percentages. Continuous variables were analyzed using Mann–Whitney


Smyrna blomfildia (Fabricius 1781)  is a sexually dimorphic species found in Mexico, Central, and South America. Males are territorial and are more vibrantly colored than females. Genome skimming by Illumina sequencing allowed the assembly of a complete circular mitochondrial genome (mitogenome) of 15,149 bp from S. blomfildia consisting of 83.9% AT nucleotides, 13 protein-coding genes, 22 tRNAs, two rRNAs, and a control region in the typical butterfly gene order. The S. blomfilda COX1 gene features an atypical start codon (CGA) while ATP6, COX1, COX2, CYTB, ND1, ND3, ND4, and ND5 display partial stop codons completed by the addition of 3’ A residues to the mRNA. Bayesian phylogenetic reconstruction places Smyrna as a member of the tribe Nymphalini and sister to a clade containing genera Araschnia, Vanessa, Polygonia, and Aglais, which differs from its classic taxonomic placement in tribe Coeini.The Blomfild’s Beauty butterfly  Smyrna blomfildia  in October 2017 that has been pinned, spread, and deposited in the Wallis Roughley Museum of Entomology, University of Manitoba  voucher WRME0507738.The e Schaus . Femalese Schaus . Females with slight modifications to the standard protocol as described in McCullagh and Marcus (S. blomfildia (Genbank MZ151338) was assembled and annotated using Geneious Prime 2021.1.1 from an SRA library of 18,400,288 paired 150 bp reads (Genbank SRA PRJNA729786) using a Baeotus beotus reference mitogenome   was also assembled and annotated using a B. beotus (MW571038) reference sequence. The rRNA repeat sequence is increasingly recognized as being very useful for phylogenetic comparisons based on nuclear markers , ATT , ATC, (ND3), and CGA, an atypical COX1 start codon that is also found in the COX1 gene of many other insects  with single-nucleotide (T) stop codons, and four protein-coding gene  with two-nucleotide (TA) stop codons completed by post-transcriptional addition of 3′ A residues. All structures of the tRNAs were verified using ARWEN v.1.2 (Laslett and Canback The S. blomfildia and 37 other mitogenomes from the family Nymphalidae. Sequences were aligned in CLUSTALX 2.1 (Thompson et al. Smyrna as a member of the tribe Nymphalini and sister to a clade containing genera Araschnia, Vanessa, Polygonia, and Aglais, confirming the findings of some previous molecular phylogenetic analyses (Wahlberg et al. Smyrna in tribe Nymphalini is also supported by larval morphological characters (Muyshondt and Muyshondt Smyrna with Baeotus beotus in the tribe Coeini based on adult morphology (Muyshondt and Muyshondt Smyrna in tribe Nymphalini.Phylogenetic reconstruction  was comp