FPR1, FPR2, and FPR3 are members of Formyl Peptides Receptors (FPRs) family belonging to the GPCR superfamily. FPR2 is a low affinity receptor for formyl peptides and it is considered the most promiscuous member of this family. Intracellular signaling cascades triggered by FPRs include the activation of different protein kinases and phosphatase, as well as tyrosine kinase receptors transactivation. Protein kinases and phosphatases act coordinately and any impairment of their activation or regulation represents one of the most common causes of several human diseases. Several phospho-sites has been identified in protein kinases and phosphatases, whose role may be to expand the repertoire of molecular mechanisms of regulation or may be necessary for fine-tuning of switch properties. We previously performed a phospho-proteomic analysis in FPR2-stimulated cells that revealed, among other things, not yet identified phospho-sites on six protein kinases and one protein phosphatase. Herein, we discuss on the selective phosphorylation of Serine/Threonine-protein kinase N2, Serine/Threonine-protein kinase PRP4 homolog, Serine/Threonine-protein kinase MARK2, Serine/Threonine-protein kinase PAK4, Serine/Threonine-protein kinase 10, Dual specificity mitogen-activated protein kinase kinase 2, and Protein phosphatase 1 regulatory subunit 14A, triggered by FPR2 stimulation. We also describe the putative FPR2-dependent signaling cascades upstream to these specific phospho-sites. Post-translational modifications (PTMs) regulate many proteins functions thanks to the addition of a functional chemical group. Phosphorylation is the most important PTMs in eukaryotic cells as it plays a critical role in regulating many eukaryotic cellular processes, including metabolism, cell cycle progression, cellular proliferation, survival, migration and differentiation . PhosphoFormyl Peptides Receptors (FPRs) belong to the GPCR superfamily and include three members  that recognize formyl peptides and/or other ligands with different range of affinity ,13. FPR2FPRs were firstly identified on myeloid cell membrane, but subsequently their expression was also observed in neuronal, endothelial and epithelial cells . Their aphox and p67phox which are required for NADPH oxidase-dependent reactive oxygen species (ROS) generation [Intracellular signaling cascades triggered by FPRs include the activation of different kinases and phosphatases, such as protein kinase C (PKC) isoforms, phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), mitogen-activated protein kinase (MAPK), p38MAPK, PTEN, PTP-PEST, and DUSP3/VHR ,18,19,20neration ,22. Furtneration ,24,25,26These signaling cascades regulate several cellular functions and their impairment significantly contributes to the development of human diseases. In fact, FPRs are functionally expressed in cells and tissues of central and peripheral nervous system, where they play a key role in the development of neurodegenerative diseases ,27, suchWe previously performed a phospho-proteomic analysis in FPR2-stimulated cells to shed light on intracellular signaling cascades triggered by this receptor. To this aim, CaLu-6 cells were serum-starved for 24 h and then stimulated for 5 min with the WKYMVm peptide, a selective FPR2 agonist . We iden2-terminal GTPase-binding region. The central domain shows a weak homology to the calcium-dependent phospholipid binding C2 domain of PKC (HR2/C2) and contains an auto-inhibitory region [2-terminus by a hinge region. The 3′-end of the COOH-terminal region of PKN1 and PKN2 is also involved in the binding to RhoA [Protein kinase C-related kinases (PKN/PRK) belong to a subfamily of AGC serine/threonine-protein kinases ,40,41. Ty region ,44,45. Ty region ,47 and cy region ,48,49. Ty region ,51. The y region  that in  to RhoA . PKN2 st to RhoA . Two hig to RhoA ,55. The  to RhoA  and may  to RhoA ,55. In P to RhoA ,57.PKN1 and PKN2 kinase activity is regulated by mTORC2-dependent phosphorylation of the turn phosphate motif . The actThe biological responses downstream to the interaction of chemoattractants with FPRs are regulated by Ras and Rho GTPases family, which induce the activation of PI3K, AKT and ERK1/2 ,66,67. PPKNs can be activated by growth factor receptors signaling through different pathways. TKR-induced PI3K activity catalyzes PIP3 synthesis which is required for mTORC2 activation and Thr958 phosphorylation in the turn phosphate motif. Furthermore, phospho-tyrosine residues of TKRs provide docking sites for SH2 and PTB domains. NCK adapter protein binds phosphorylated Epidermal Growth Factor Receptor (EGFR), Platelet-Derived Growth Factor Receptor (PDGFR), Vascular Endothelial Growth Factor Receptor (VEGFR) via its SH2 domain and is a mediator of growth factor-induced signal transduction ,70,71. PPKN2 is one the first identified effector proteins of Rho  and playSchizosaccharomyces pombe, but its expression has been subsequently observed in a variety of human tissues [2-terminus, which is characterized by basic aminoacids repeats, able to act as a NLS, and Arg/Ser (RS) domains, frequently found in pre-mRNA splicing factors whose deletion induces the cytosolic localization of the protein [Cdk, MAPK, GSK, and cdc2-like kinase (Clk) protein families [PRP4 was originally isolated from  tissues . Human P protein . PRP4 be protein ,87 and, families . PRP4 befamilies . MAPK fafamilies . The regfamilies ,91. Thr3families .847PY849 motif in the subdomain VIII of PRP4 is not essential for kinase activity which requires, at least partially, the autophosphorylation on Ser and Tyr residues [PRP4 is involved in pre-mRNA splicing. In fact, it is able to phosphorylate the RS domain of the mammalian splicing factor ASF/SF2 , which bresidues . Neverthresidues . PRP4 isresidues  and playresidues . Previouresidues . We specMAP-microtubule affinity-regulating kinases (MARKs) were discovered for their ability to phosphorylate tau and related microtubule-associated proteins (MAPs) on Ser residues located within the KXGS motifs. Phosphorylation of tau results in the detachment from microtubules and in the impairment of microtubule-based intracellular transport ,99. In C2-terminal header, (ii) a catalytic kinase domain, (iii) a linker, (iv) a UBA domain, (v) a spacer, and (vi) a tail domain [The alpha subunits of AMPK belong to a family of 12 related kinases , namely BRSK1, BRSK2, PAR-1c/MARK1, PAR-1b/MARK2, PAR-1a/MARK3, PAR-1d/MARK4, MELK, NUAK1/ARK5, NUAK2/SNARK, SIK1, SIK2/QIK and SIK3/QSK . PAR-1c/l domain . The roll domain .Interestingly, both PAR-1b/MARK2 and AMPK are regulators of metabolism and show overlapping functions in vivo ,107. In In addition to tau and MAPs, MARKs phosphorylate several substrates in their binding sites, such as mitogen-activated protein kinase scaffolding protein KSR1, tyrosine phosphatase PTPH1, cell cycle-regulating phosphatase Cdc25, class IIa histone deacetylases and plakophilin. Such phosphorylations enhance the binding of these proteins to 14-3-3 modifying their subcellular localization and affecting regulatory pathways ,114.MARK2 is activated by phosphorylation of a conserved Thr208 in the activation loop (T loop) located within the catalytic domain. MARKK/TAO-1 and the tumor suppressor Liver kinase B1 (LKB1) in complex with STRAD and MO25 phosphorylate MARK2 at this site ,116. On MARKK/TAO-1 is regulated by Ras GTPases family ,123 and 2-terminal PBD (p21-GTPase-binding domain), also known as a Cdc42 Rac Interactive Binding (CRIB) domain [2-terminal PBD sequence, there is considerable homology within the PBD between all PAK family members. PAK1-3 bind to Rac and Cdc42, whereas PAK4-6 binds preferentially Cdc42. None of the PAK family members bind to the Rho isoforms RhoA, RhoB, and RhoC [The p21-activated kinases (PAKs) belong to the Ser/Thr kinases family which is composed of six members distributed into a group I  and a group II . The PAK family proteins show a similar structure that can be divided into three main domains: an NH) domain , a centr) domain . Even thand RhoC . The cenand RhoC , and an and RhoC .2-terminal regions of PAK4 and PAK5 can interfere with kinase activity. In fact, removal of the PBD enhances PAK4 and PAK5 kinase activity [2-terminal fragment able to inhibit kinase activity has not been identified in PAK4. The kinase activity of PAK4 can be inhibited by the PI3K inhibitor LY294002 but the mechanism of regulation of PAK4 kinase activity by PI3K is still unclear [2-terminal region, and a nuclear export sequence [The activation mechanisms of groups I and II of PAKs are different and the relative regulatory domains are structurally distinct . The kinactivity ,138, sug unclear ,135. PAKsequence ,132.Therefore, PAK4 is mainly activated by cMet-induced PI3K signaling. Noteworthy, PAK4 and the p85 α-subunit of PI3K specifically interact  and the Previously, we showed that FPR2 stimulation by WKYMVm induces the trans-phosphorylation of Y1313/Y1349/Y1356 residues of c-Met and elicits some of the molecular responses triggered by c-Met/HGF binding, such as STAT3, phospholipase C (PLC)-γ1/PKCα and PI3K/Akt pathways ,143. We 2-terminus, which shows similarity to that of the STE20 family of serine/threonine kinases involved in regulating MAPK cascades, and a long coiled-coil structure at the COOH-terminus. The two domains are separated by a proline-rich region. In yeast STE20 is a component of the MAP kinase cascade required to transmit the pheromone signal from Gβγ subunits to downstream components [In mice, lymphocyte-oriented kinase (LOK) is a member of the STE20 family characterized by serine/threonine kinase activity, whose expression is restricted mostly to lymphoid cells . It consmponents . STE20 pmponents . Severalmponents ,158.STK10 is the human homolog of LOK. It shows high similarity to LOK with a 98% and 93% of identity in the kinase domains and in the coiled-coil regions, respectively . STK10 sSTK10 is a key kinase for tumor progression  and siRNSTK10 phosphorylates ezrin, radixin and moesin (ERM), which mediate linkage of actin cytoskeleton to plasma membrane  and contThe upstream signaling cascades regulating STK10 kinase activity remain to be elucidated, although STK10 catalytic activity seems to depend on dimerization of the kinase domain and, in turn, on STK10 trans-phosphorylation . InteresWe found that Ser438 and Thr952 residues of STK10 are phosphorylated in FPR2-stimulated cells , in humaDual specificity mitogen-activated protein kinase, kinase 2 (MAP2K2) is also known as CFC4, or MAPKK2, or MEK2 , or MKK2 (MAP kinase kinase 2), or PRKMK2. MEK1 and MEK2 are ubiquitously expressed and are encoded by two distinct genes located at different chromosomes . The MAPphox, p47phox and p67phox and their translocation to the membrane, followed by their interaction with cytochrome b558 [phox on Ser and Thr residues [phox phosphorylation [phox phosphorylation and membrane translocation, as well as NADPH-dependent superoxide production [NADPH oxidase activation requires the phosphorylation of the cytosolic components p40ome b558 ,195,196.residues ,198,199 rylation ,201, sugoduction ,22.In a typical signaling pathway triggered by TKRs, the phosphorylated tyrosine residues of the TKRs act as docking sites for several proteins, including Shc, Grb2 and SOS that link the activated TKR to Ras-signaling pathway. The GTP-bound Ras induces the translocation of the Ser/Thr kinase Raf-1 to the plasma membrane, where it is activated by multiple phosphorylation events ,203. Raf2- and COOH-terminal tails and a central PHIN domain that consists of 86 residues [2-terminal tail domain, absent in CPI-17 [PPP1R14A is also known as CPI-17, CPI17 or PPP1NL. Its structure shows intrinsically disordered NHresidues ,209,210.residues . Three Cn CPI-17 .CPI-17 is expressed predominantly in smooth muscles where it plays crucial regulatory functions ,213,214.2+ and an initial rapid MLCP phosphorylation. On the other hand, ROCK stimulates the Ca2+-independent phosphorylation of CPI-17 in the subsequent sustained phase, in parallel with a force generation [2-terminal tail [2+/calmodulin-dependent protein kinase II phosphorylates the Ser130 residue at the COOH-terminal tail of CPI-17 [A major kinase for GPCR-induced CPI-17 phosphorylation is PKC which is activated by the PLCβ-produced signaling messenger diacylglycerol (DAG). It phosphorylates CPI-17 at Thr38 residue that directly docks at the active site of MLCP, thereby inhibiting its activity and promoting an increase of phosphorylation of myosin and of other MLCP substrates ,220,221.neration . CPI-17 neration . CPI-17 neration . Sequencnal tail . In addinal tail ,228,229,f CPI-17 .We observed that FPR2 stimulation induces the phosphorylation of Ser16 and Ser26 residues of CPI-17 . PhosphoTherefore, CPI-17 activity is regulated by PKC and ROCK and several GPCR agonists induce PKC- and ROCK-dependent phosphorylation of CPI-17 at Thr38 residue ,226,230.FPR2 is a versatile transmembrane protein activated by an array of agonists, which include structurally unrelated lipids and peptide ligands, resulting in different intracellular responses. The ability of FPR2 to mediate a multitude of physio-pathological processes can be traced to the different receptor domains used by distinct agonists. FPR2 represents a promising therapeutic target in several human diseases, but further studies are required to fully understand molecular mechanisms responsible for PKs and PPs activation upon stimulation with different agonists.PKs mediate a network of highly complex signals. The understanding of the regulatory functions of kinases may represents a valuable tool to identify more effective therapies against several human diseases. In fact, many drug kinase inhibitors are used in the treatment of different types of cancer and phospho-proteomics represents an important analytical strategy to identify new phospho-sites and phospho-proteins, and in turn to develop new drugs in tumor biology.Many proteins are phosphorylated, even though phosphorylation is only one of several types of reversible covalent PTMs, which also include methylation, acetylation, sumoylation, and ubiquitination. In phospho-proteins, only a limited subset of phosphorylated sites is regulated. Furthermore, the observation that single phospho-sites are differently regulated suggests that proteins function as platforms to integrate multiple incoming stimuli. This integration of signals could act in an independent- or dependent manner. In the first case, the phosphorylation of each site occurs independently from the others, whereas in the second case a “priming site” is necessary for additional phosphorylation events. Inactivating mutations of PKs have been identified only in a small number of human cancers, indicating that modifications in kinase activity by phosphorylation are among the main causes of disease progression, and that phospho-proteomic screens could be useful to monitor cancer development and to identify new potential therapeutic targets.


Polaribacter, Pseudoalteromonas and HTCC2207, responded positively to decreased salinity in both the bay transect and experiments. The relative abundance of these bacteria slightly decreased, but their functional activity was maintained and increased the over time in the dilution experiments. However, while ice bacteria, such as Flavobacterium and Polaromonas, tolerated the increased salinity after mixing with seawater, their gene expression decreased considerably. We suggest that these bacterial taxa could be defined as sentinels of freshening events in the Antarctic coastal system. Furthermore, these results suggest that a significant portion of the microbial community is resilient and can adapt to disturbances, such as freshening due to the warming effect of climate change in Antarctica.Current warming in the Western Antarctic Peninsula (WAP) has multiple effects on the marine ecosystem, modifying the trophic web and the nutrient regime. In this study, the effect of decreased surface salinity on the marine microbial community as a consequence of freshening from nearby glaciers was investigated in Chile Bay, Greenwich Island, WAP. In the summer of 2016, samples were collected from glacier ice and transects along the bay for 16S rRNA gene sequencing, while in situ dilution experiments were conducted and analyzed using 16S rRNA gene sequencing and metatranscriptomic analysis. The results reveal that certain common seawater genera, such as  As a consequence of climate change, polar regions are the most radically changing places on the planet ,2. Over Roseobacter clade and Gammaproteobacteria , photoheterotrophs , and aerobic anoxygenic phototrophs , while autotrophic carbon assimilation is mainly driven by small oxygenic photoautotrophic eukaryotes [a (Chl a) concentration during summer blooms, where Gammaproteobacteria , Alphaproteobacteria  and Bacteroidetes  have the largest relative abundances and activity [Sulfitobacter and some Rhodobacterales  clades of Alphaproteobacteria, together with some Gammaproteobacteria , seem to be prevalent during both the summer and winter seasons [Surface bacterioplankton communities show considerable variability between different areas of Antarctica. However, Bacteroidetes, Gammaproteobacteria, Alphaproteobacteria and Actinobacteria are generally the dominant bacterial taxa in the deep subsurface shelf waters throughout the year ,23,24,25karyotes ,24,26,27 seasons ,28,29.Climate change will likely result in more ice melting and this, in turn, will increase water column stratification, which has been shown to have an impact on micro-eukaryotic and bacterial community composition and primary production ,30,31. AIn the present study, the microbial community composition along a surface salinity gradient in the coastal waters and surrounding glacier ice at Chile Bay (Greenwich Island) in the WAP were analyzed using 16S rRNA gene sequencing. Additionally, in situ dilution experiments were carried out, with the most active bacteria and their metabolic capacities evaluated using metatranscriptomics. These approaches generated the first example of comprehensive data that explores the effect of (i) ice–freshwater discharge and (ii) ice–microorganism transport into coastal systems due to glacial melt and consequent freshening. This information contributes to the understanding of how ecological shifts in response to specific factors such as salinity will affect specific members of the microbial community. Ultimately, this will provide information as to how the accelerating reduction of ice cover and consequent freshening of coastal waters will potentially influence the microbial communities in coastal Antarctic waters.−3) at distant stations P1 and P3 were obtained using a SeaBird19 CTD. Seawater (20 L) was collected at the three stations using a manual pump on board a Zodiac boat. The water samples were directly deposited into previously cleaned bottles (10% HCl) and then transported to the laboratory at the Chilean station Captain Arturo Prat (Instituto Antártico Chileno), Chile Bay. Triplicate seawater subsamples for nutrient analysis were filtered with 0.7 µm GF/F glass fiber filters, placed in 15 mL polypropylene tubes and stored at −20 °C until laboratory analysis. The final concentration of nitrite (µM) (detection limit 0.05 μM), nitrate (µM) (detection limit 0.05 μM), phosphate (µM) (detection limit 0.02 μM) and silicate (µM) (detection limit 0.65 μM) were obtained by standard colorimetric assays [a concentration was determined in triplicate by collecting 1 L of seawater on a 0.7 µm GF/F glass fiber filter. Next, the contents of the frozen filters were extracted with acetone (90%) and then the extract was measured fluorometrically [a and nucleic acids were collected from the melted ice water using the same protocol described for seawater.Chile Bay is located on the north side of Greenwich Island in the South Shetland Islands, WAP . This Antarctic expedition was undertaken from 18th February to 4th March 2016. Seawater samples were collected from the surface layer (2 m depth) at three different stations across an in situ salinity gradient in the bay . Stationc assays , and detc assays  which cotrically . Small p-a (1 L) at t1 (24 h) and t7 (7 d). To analyze the microbial community diversity changes, 3 L subsamples were taken at t1 and t7  from all bottles to extract DNA for subsequent 16S rRNA gene sequencing. An additional 3 L were collected from each bottle at t7 for RNA extraction and metatranscriptomic analysis to determine the most active bacterial components and metabolic changes potentially related to the salinity shift perturbation.Dilution experiments were conducted to simulate decreased salinity due to glacier ice melt. Prefiltered (150 µm polyester mesh to discharge large microorganisms) surface seawater (SW) from station P3 was mixed with melted ICE in clear polycarbonate bottles (20 L) to reduce the salinity from 34.2 to 24.0, which represents the typical salinity found in Subantarctic continental areas surrounded by glaciers and rivers, such as those in Chilean Patagonia. Two different microcosm conditions were performed: SW+ICE  and SW+ICE-F  . AdditioTM) was added to each filter before storage. All filters (both DNA and RNA) were immediately frozen in liquid nitrogen until further analysis in the laboratory. The same procedure was performed for the melted glacier ice samples and the dilution microcosms experiments.For in situ microbial biomass concentration and subsequent DNA/RNA extractions, seawater samples were collected along the salinity gradient at stations P1, P2 and P3 in Chile Bay, as well as for the melted glacier ice collected near the bay. For each seawater sample, 3 L of prefiltered seawater and melted glacier ice (150 µm polyesters mesh) was passed through a 0.22 µm pore size PES Sterivex filter  using a Cole Palmer peristaltic pump system (600 rpm). To preserve the integrity of the RNA, 1 mL of RNAlater RNA Stabilization Solution  was added; then, the mixture was subjected twice to 20 s of bead-beating. An RNA Clean & Concentrator kit  was then used according to the instructions. Finally, the quality and quantity of the total extracted nucleic acids were checked with a Qubit fluorometer (Invitrogen) and a dsDNA-BR Assay kit for DNA or an RNA-BR Assay kit for RNA. The integrity was checked via 0.8% agarose gel (DNAse/RNAse-free) electrophoresis.For RNA extractions, each RNA filter was cleaned of RNAlater by successive washes with sterilized ddHwww.earthmicrobiome.org/protocols-and-standards). The V4-V5 hypervariable region of the 16S rRNA gene was amplified by PCR using the bacterial–archaeal primer pairs 515F (5′GTGYCAGCMGCCGCGGTAA 3′) and 926R (5′CCGYCAATTYMTTTRAGTTT3′) [16S rRNA gene i-Tag sequencing was performed on DNA samples from stations P1 (4), P2 (1) and P3 (6) during 18th February to 4th March 2016, and on samples t1 and t7 and the controls  from the dilution microcosms experiments. Amplification and sequencing of the 16S rRNA gene was performed according to the Earth Microbiome Project protocols (AGTTT3′) . The amphttp://phytoref.sb-roscoff.fr, downloaded on 8 October 2020). Both assignations were done with the classify-consensus-vsearch plugin implemented in Qiime2. Reads classified as mitochondria (1.8%) were discarded. The final Bacteria/Archaea table was composed of 339,220 reads distributed into 893 ASVs across the 19 samples . The plastid table was composed of 60,062 reads distributed into 76 ASVs across the same 19 samples.Processing of the 16S rRNA gene sequences was performed with the QIIME2 package v.2018.11  and the Between 700 ng and 1 µg of clean total RNA extracted from the control (SW and ICE), SW+ICE and SW+ICE-F microcosms experiments at the final time (t7) were sequenced for metatranscriptomic analysis via Illumina Hi-Seq technology . Briefly, the total RNA was processed using standard procedures and depleted of rRNA using RiboZero. The RNA-Seq libraries were prepped using NEXTflex Rapid Directional RNA-Seq Kit from Bioo Scientific. The quality of the metatranscriptomic raw reads was assessed with FastQC . The sequences were quality filtered using Cutadapt . The firThe principal genes involved in energetic metabolism associated with carbon fixation and nitrogen assimilation were searched for in the dilution microcosm experiments.Polaromonas, Polaribacter and Flavobacterium proteins where specifically obtained from several genomes recovered from the Genome Taxonomy Database , due to their low representation in the TARA Oceans MAGs [Prodigal  . Finally, the KOs were organized into pathways using information from the KEGG website. The KO and Pfam accession IDs for the carbon and nitrogen cycles were used to analyze these specific pathways in this study and are listed in Annex 1. All scripts used in this work are available upon request. This data has been deposited in the NCBI database under accession number PRJNA663269 and sample accession numbers SAMN16124624 to SAMN16124642.To obtain the microbial gene transcriptional levels, non-rRNA metatranscriptomic reads obtained from microcosms  at the t7 final time were recruited to our custom protein database using DIAMOND  with an cesscore . As eggNcesscore . The finDuring the late summer (February) of 2016, three different stations  along an in situ surface salinity gradient (32.5 to 34.2) were sampled in Chile Bay, WAP a. AdditiSurface seawater temperature remained around 2.0 °C along the transect and decreased with depth down to 1.6 °C b. A surfa concentrations were also low in the glacier ice at around 0.291 ± 0.02 mg m−3, while they were one order of magnitude higher in the seawater, with an average value of 3.65 mg m−3  of incubation, the SW control showed a high decrease of nitrite (33%), nitrate (67%), phosphate (55%) and silicate (44%). However, in the SW+ICE microcosms, the most notably decreased nutrients were nitrate (32%) and phosphate (34%), and in the SW+ICE-F the same nutrients decreased 23% and 30%, respectively (a at t7 increased 14-fold in SW (23.5 mg m−3) and SW+ICE-F (16.1 mg m−3), and 18-fold in SW+ICE (17.1 mg m−3), denoting high biological activity inside the microcosms after 7 days of incubation , and Sphingobacteriales (22.3%), particularly the genus Pedobacter (20.9%). Burkholderiaceae (22.1%), mostly represented by Polaromonas (9.6%), was the most abundant proteobacterial group. The seawater microbial community was also dominated by Bacteroidetes  and Proteobacteria . However, taxonomic affiliations showed Polaribacter  as the most abundant Bacteroidetes. Within Proteobacteria, Alphaproteobacteria , especially Sulfitobacter, was the dominant group rather than Gammaproteobacteria . The relative abundance of Sulfitobacter  decreased slightly from P1 (19.5%) to P3 (16.6%), showing a similar pattern as Polaribacter  increased with time. The glacier ice community dominated the SW+ICE experiment at t1  and decreased to 14.2% at the final time and Pedobacter showed minor changes from t1 to t7  (Polaribacter (2.2%) and Sulfitobacter (4.5%), was detected at t1, but these community members increased by t7, reaching 6.8% and 14.3%, respectively. As expected for the SW+ICE-F experiment  and t7 (17.3%) in the SW+ICE-F experiment. In turn, Sulfitobacter increased by 24% at t7 (32.4%) with respect to the initial time (26.2% (t1))  b. A low periment b, the se2% (t1)) b.Flavobacterium and Polaromonas were the most active members, while Pedobacter did not exhibit much activity. In the three seawater experiments, Polaribacter was the most active genus at the final time, showing 14% lower activity in the dilution experiments. Conversely, Pseudoalteromonas showed higher expression levels in the dilution experiments. Two other bacteria showed opposite patterns: HTCC2207 exhibited relatively high expression in contrast to its low abundance in all SW-containing incubations, while Sulfitobacter had low activity  to live when glacier ice was mixed with SW (SW+ICE). The functions that most decreased were ribosome components (40-fold), RNA polymerase (11-fold), terpenoid backbone biosynthesis and homologous recombination activities. Flavobacterium, another glacier ice representative, in general showed a 24-fold decrease in the activity of metabolic functions in SW+ICE relative to the ICE control; a 40-fold decrease in expression of genes for ABC transporters, ribosome components and RNA degradation; and a more than 90-fold decrease in terpenoid backbone biosynthesis activity  region has undergone significant changes in temperature and seasonal ice dynamics, with substantial impacts on the regional ecosystem, ocean chemistry and hydrographic properties ,7,54. AnChile Bay in the WAP is considered a highly productive system during the austral summer, exhibiting seasonal changes in the bacterial and eukaryotic communities ,25. Altha concentrations (up to 4 mg m−3) as those previously reported for the late summer of 2014 [Polaribacter exhibited a slight reduction in their abundance further offshore where the seawater is more saline; however, Polaribacter was one of the most dominant members in terms of relative abundance at near-glacier sites where the salinity is lower, suggesting high adaptation to freshening. Polaribacter has been previously suggested to originate from melting sea ice in the Arctic and Antarctica [Polaribacter abundances found in our seawater samples during the summer of 2016, while we only detected a low abundance (<5%) in the glacier ice samples. Another dominant seawater taxon was Sulfitobacter, a Roseobacter-clade member that is considered a predominant bacterioplankton in the Antarctic Peninsula [Polaribacter, the abundance of this bacterium slightly decreased as the transect became saltier. Although the prevalence of these two genera has been described as seasonal and variable between poles [In order to understand the response of marine microbial communities in Chile Bay to natural freshwater input from glacier melting, we analyzed changes in the relative abundances of marine bacteria along a transect from the glacier towards offshore marine waters of the bay during the summer of 2016. Our study demonstrates the presence of a diatom phytoplankton bloom during the 2016 summer sampling, with similar Chl  of 2014 . We demotarctica ,56, whertarctica . This mieninsula , and a ceninsula ,60,61. Sen poles ,28,62, iFlavobacterium, Pedobacter and Polaromonas, which have also been widely reported in other glacier ice ecosystems [Flavobacterium, the most dominant genus, is widespread in nature and has been isolated from many freshwater and soil habitats, including polar systems. This bacterium plays a crucial role in the remineralization processes and exhibits strong macromolecular hydrolytic capabilities [Moreover, the glacier ice microbial community near Chile Bay exhibited dominance of the phyla Proteobacteria and Bacteroidetes, as previously reported for other glacier samples, such as those from the Svalbard Archipelago . Here, tosystems ,64,65. Fbilities . BesidesAltogether, our results suggest that major seawater representatives show subtle adaptation to the decreased surface salinity in Chile Bay due to local glacier melting. These findings led us to connect the bacterial composition and abundances of these two environments in order to understand how the bacterial community changes when faced with natural freshening events. In this way, Chile bay is an ideal natural coastal site in Antarctic marine waters to test how climate change is affecting the WAP in relation to prior effects on terrestrial environments, such as glacier melting. Microbial sentinels, for those types of effects, are now needed to fully understand the impact that climate change is having in the region.In order to confirm some of our previous mentioned results obtained from natural waters of Chile Bay, dilution experiments with Chile Bay marine waters were performed. It is a fact that the WAP has been subject to periods of warming surface temperatures and that enhanced mass loss from melting glaciers has triggered consequences for the functional characteristics of the whole ecosystem ,68. HerePolaribacter, which live and subsequently thrive in terms of relative abundance, maintaining important gene expression after seven days in the dilution microcosms  in response to dilution  could be the eukaryote Phaeocystis antarctica, which is a strong DMSP producer in low salinity under freezing conditions [Phaeocystis antarctica was present in Chile Bay in 2016  when facing dilution in the microcosms, denoting good performance of the Calvin cycle and photosynthesis , as observed in the ice brine of the Arctic [Additionally, two seawater taxa that were hardly detected in the 16S rRNA data showed increased gene expression after seven days in the diluted incubations. The first was HTCC2207 , which exhibited increased gene expression (3%) in the diluted experiments compared to the SW control. This Gammaproteobacteria is an important global component of the marine sulfur cycle and has been described as the most relatively abundant bacterioplankton group of the northern Antarctic Peninsula . The secfixation . This imtivities . Gene exe Arctic ,83, coulFlavobacterium spp., decreased in abundance and gene expression by the end of the 7-day experiment. Our results show that carbon metabolism experienced a notable decrease in expression, together with ABC transporters, protein machinery and RNA degradation. As Flavobacterium is a widespread taxon, including in polar systems, our results indicate that, despite exhibiting lower expression of important metabolic pathways during the experiment, it is able to maintain certain activity while potentially tolerating saltier water. Similarly, Polaromonas showed lower activity of the same metabolic pathways; however, this only represented a 2-fold decrease compared to the >10-fold decrease seen for Flavobacterium  or tolerance accompanied by a rapid decrease in gene expression . Moreover, the positive behavior of eukaryotes during salt dilution could help us to model these events. These results also provide information about the maintenance of the primary and secondary productivity of Chile Bay and, possibly, all coastal waters of the WAP, where freshening events occur throughout the summer. This is of great importance for all Antarctic coastal ecosystems since potential sentinels are the main microorganisms involved in carbon and nitrogen biogeochemical cycles.Additionally, through these dilution experiments, several relevant and common marine genera (bacteria and eukaryotes) reacted positively by increasing the expression of hundreds of genes in response to salt dilution. During salinity changes in Chile Bay due to glacier freshening, our results strongly suggest bacterial functional resilience (e.g.,


Furthermore, the docking analysis revealed that the ACE inhibition of GHIITVAR was mainly attributed to forming very strong hydrogen bonds with the active sites of ACE. These results identify sesame protein as a rich source of ACE inhibitory peptides and further indicate that GHIITVAR has the potential for development of new functional foods.The aim of this study was to isolate and identify angiotensin I-converting enzyme (ACE) inhibitory peptides from sesame protein through simulated gastrointestinal digestion in vitro, and to explore the underlying mechanisms by molecular docking. The sesame protein was enzymatically hydrolyzed by pepsin, trypsin, and α-chymotrypsin. The degree of hydrolysis (DH) and peptide yield increased with the increase of digest time. Moreover, ACE inhibitory activity was enhanced after digestion. The sesame protein digestive solution (SPDS) was purified by ultrafiltration through different molecular weight cut-off (MWCO) membranes and SPDS-VII (< 3 kDa) had the strongest ACE inhibition. SPDS-VII was further purified by NGC Quest™ 10 Plus Chromatography System and finally 11 peptides were identified by Nano UHPLC-ESI-MS/MS (nano ultra-high performance liquid chromatography-electrospray ionization mass spectrometry/mass spectrometry) from peak 4. The peptide GHIITVAR from 11S globulin displayed the strongest ACE inhibitory activity (IC Hypertension is one of the diseases with the highest mortality in the world, and it is the main pathogenesis factor of coronary heart disease, stroke, and heart and kidney failure ,2,3. AccRelevant studies have shown that human blood pressure is regulated by many factors, among which renin-angiotensin system (RAS) and kallikrein kinin system (KKS) are the main ways to control the stability of blood pressure. ACE is a dipeptide carboxyl metalloproteinase which plays a key role in these two systems. In the RAS system, ACE can convert the inactive angiotensin I into angiotensin II, which has the function of constricting vascular smooth muscle, thus, increases the blood pressure. Meanwhile, ACE can inactivate the vasodilator bradykinin in the KKS system, leading to vasoconstriction, which causes an increase in the blood pressure . CurrentBioactive peptides are small fragments of food protein, mostly composed of 2–20 amino acid residues. They have the characteristics of easy absorption, low sensitivity and good solubility. Meanwhile, they have the function of nutrition and physiological regulation, which have obvious effects on the conditioning and treatment of modern chronic diseases and sub-health state. During processing, enzymatic hydrolysis, fermentation, or gastrointestinal digestion, foods can release bioactive peptides ,9 which Sesamum indicum L.) is one of the important oil seed crops worldwide and it is widely used in food, health care, and medical applications because of its high nutritional value [Sesame  represents the extent of protein degradation, which has been widely used in hydrolysis efficiency assessments. As shown in 50 values inhibiting ACE activity were calculated from the results of a series of concentrations. As shown in 50 values of these fractions were 35.143 ± 1.122, 15.066 ± 0.042, 9.146 ± 0.005, 6.108 ± 0.001, 5.106 ± 0.003, 4.583 ± 0.003, 2.720 ± 0.003 μg/mL, respectively. A lower IC50 value corresponds to a stronger inhibitory activity. SPDS-VII with molecular weight <3kDa exhibited the strongest ACE inhibitory activity with the lowest IC50 value among these fractions, indicating that lower molecular weight peptides or small molecules showed stronger ACE inhibition. This result was in agreement with previous studies reporting that most ACE inhibitory peptides derived from food protein were generally short sequences containing 2–20 amino acids, or the hydrolyzates were generally filtered through a 3 kDa membrane [It is well known that molecular weights of peptide fragments are crucial for their biological activities. Ultrafiltration is a membrane separation technique that is of great use in the concentration, purification, fractionation, and clarification of solutes. It has high throughput of product, low operational cost, energy saving potential, and ease of equipment cleaning. In the separation of polypeptides, ultrafiltration can effectively remove insoluble substrates, large molecular weight proteins and peptides, and obtain bioactive peptides with smaller molecular weight ,24. The membrane ,26. In amembrane .50 value of (2.847 ± 0.045) μg/mL, peak 2 with the retention time from 6.52 to 7.12 min exhibited an IC50 value of (1.421 ± 0.035) μg/mL. The IC50 value of peak 3 with a retention time from 7.12 to 7.42 min was (1.838 ± 0.026) μg/mL, peak 4 with a retention time from 7.49 to 8.27 min was (0.558 ± 0.003) μg/mL and peak 5 with a retention time from 8.27 to 10.80 min was (0.757 ± 0.014) μg/mL. These results demonstrated that peak 4 possessed the most potent ACE inhibitory activity among the five peaks and thus was selected for further identification.To enhance the ACE inhibition, SPDS-VII was further purified by NGC Quest™ 10 Plus Chromatography System. It is an effective way to purify proteins or peptides and collect them in the preparation phase. The chromatogram is shown in Sesamum indicum L. were found in Uniprot database (http://www.uniprot.org/). In this study, a total of 11 peptides were identified by analysis of the MS/MS spectrum  from sesame protein hydrolyzed by alcalase [The peptides from peak 4 were identified by Nano UHPLC-ESI-MS/MS. The amino acid sequences from trum see . The seqalcalase , suggestAccording to previous literature reports, most of the proteins present in sesame seeds are storage proteins composed of globulins (67.3%), albumins (8.6%), prolamines (1.4%), and glutelins (6.9%), of which sesame 11S globulins includes 11S globulin seed storage protein 2 precursor, 11S globulin isoform 3, and 11S globulin isoform 4 ,29. As s50 = 54.79 ± 0.37 μM) and FMPGVPGPIQR (IC50 = 11.08 ± 0.15 μM) in ACE inhibition further supported the above conclusion. Nevertheless, there were some exceptions, for example, the peptide EN and EVD from whey protein and the peptide CRQNTLGHNTQTSIAQ from Stichopus horrens do not have any reported specific amino acid at the C-terminal [It is known that the ACE inhibition is strongly influenced by the type of amino acid composition of peptides. A series of studies have figured out that binding to ACE is influenced by hydrophobic amino acid residues (aromatic or branched chain) at three positions from the C-terminus of the peptide ,31. Someterminal ,35. Thus50 value of 3.60 ± 0.10 μM among the 11 peptides. The inhibition potential of GHIITVAR was still less than the synthetic ACE inhibitor Captopril (IC50 = 0.0071 μM). GHIITVAR contained a hydrophobic amino acid Arg at the C-terminus and two aliphatic amino acids  at the antepenultimate and penultimate positions, respectively. Besides, the presence of two aliphatic amino acids Ile may contribute to ACE inhibitory activity. However, the IC50 value of the peptide HIITLGR was 74.65 ± 0.13 μM, which further confirmed the fact that the presence of hydrophobic amino acids, especially those with aliphatic chains such as Gly at the N-terminus might enhance the ACE inhibition. There were nine peptides  containing Arg in this study . Arg is https://web.expasy.org/peptide_cutter/). From these results mentioned above, it could be concluded that the addition of trypsin and α-chymotrypsin significantly increase the exposure of active sites and thus increase ACE inhibitory activity. This was also consistent with the results in In addition, the cleavage sites of pepsin were hydrophobic amino acids such as Phe, Trp, Leu, and Tyr. Trypsin preferentially cleaved at Arg and Lys and α-chymotrypsin preferentially cleaved at Trp, Tyr, and Phe. The cleavage site specificity of pepsin, trypsin, and α-chymotrypsin was found in bioinformatics tools  was docked again with the prepared ACE. Taking GHIITVAR as an example, the C scores of the 20 conformations were different, but the Total scores gradually decreased . Therefo2+) in its active site that coordinates with His383, His387, and Glu411 [2+. Similarly, the peptide HIITLGR displayed in 2+. These results revealed that GHIITVAR could effectively interact with the active sites of ACE and might explain why this peptide exhibited exceptional ACE inhibition.Moreover, molecular modeling of the interaction between the peptides and ACE was further analyzed to explain the difference in the ACE inhibitory activities by using MOE. d Glu411 ,40,41. Td Glu411 . As showTypically, bioactive peptides are delivered via oral administration. Therefore, it is crucial to study their changes during gastrointestinal digestion. These peptides by enzymatic hydrolysis of exogenous proteases may be easily metabolized or further hydrolyzed in the digestive system, which may result in reduced or increased potency, or to a complete loss of activity . In the ACE inhibitory tripeptide generally requires a hydrophobic, a positively charged, and an aromatic amino acid residue at the N-terminal, middle, and C-terminal, respectively. Indeed, exact length of the C-terminal region to efficiently inhibit ACE activity remains unknown. Long-chain peptides could not always be speculated from that of their C-terminal tripeptide residues, implying the importance of other positions such as the fourth and so on . García-http://www.uwm.edu.pl/biochemia/index.php/pl/biopep) revealed that it is the first time the peptides with potent ACE inhibitory activity extracted from Sesamum indicum L were obtained. Therefore, sesame protein would be an attractive raw material generating ACE inhibitory peptides for controlling hypertension. Of course, further studies on the in vivo effects and constructing a three-dimensional quantitative structure–activity relationship (3D-QSAR) model still needs further investigation. Additionally, searching against the BIOPEP database (Sesamum indicum L.) seeds were purchased from China Oil and Foodstuffs Corporation . Angiotensin Converting Enzyme (ACE) from rabbit lung, N-Hippuryl-His-Leu (HHL) hydrate power , trifluoroacetic acid (TFA), 2,4,6 trinitrobenzene sulfonic acid (TNBS), and O-phthaldialdehyde (OPA), were purchased from Sigma-Aldrich . Pepsin (3000-3500 NFU/g), trypsin (250.N.F.U/mg), and α-chymotrypsin (1200 u/mg) were obtained from Solarbio Science and Technology Co., Ltd. . Nanofiltration and ultrafiltration membranes were purchased from Rising Sun Membrane Technology Co., Ltd. . All other reagents used in this study were of analytical grade.White sesame , SPDS-II (molecular weight between 50 and 100 kDa), SPDS-III (molecular weight between 30 and 50 kDa), SPDS-IV (molecular weight between 10 and 30 kDa), SPDS-V (molecular weight between 5 and 10 kDa), SPDS-VI (molecular weight between 3 and 5 kDa), and SPDS-VII (molecular weight below 3 kDa). These fractions were evaporated and lyophilized for subsequent ACE inhibitory study.v/v) TFA) and mobile phase B (methanol containing 0.1% (v/v) TFA). The sample was separated by a linear gradient elution from 55% to 60% of mobile phase A at a flow rate of 7.5 mL/min. The absorbance was monitored at 220 nm with a UV detector. Each chromatographic peak was pooled by repeated injection with corresponding test tube respectively through the fraction collector and lyophilized to determine the ACE inhibitory activity. The screened fraction with the strongest ACE inhibitory activity was purified by NGC Quest™ 10 Plus Chromatography System. First, 0.5 mL of the sample was filtered through a 0.22 µm membrane and injected into the system  with a Shim-pack GIS C18 preparative column . The experiment was executed by using mobile phase A . The mobile phase A was 2% acetonitrile, 0.1% formic acid, and the mobile phase B was 98% acetonitrile, 0.1% formic acid. The separation was carried out at a flow rate of 300 NL/min through the following gradient elution: 0–5 min, 5% mobile phase B; 5–45 min, mobile phase B linearly increased from 5% to 25%; 45–50 min, mobile phase B increased from 25% to 35%; 50–52 min, mobile phase B increased from 35% to 80%; 52–54 min, 80% mobile phase B; and 54–60 min, 5% mobile phase B. The eluted peak composition with the strongest ACE inhibitory activity was firstly performed by Thermo Ultimate 3000 UHPLC . The sample was enriched and salt-removed in a trap column, and then was injected onto the CThen mass analysis was performed on a Thermo Q-Exactive HF tandem mass spectrometer  after nano ESI ionization. The ion source voltage was set to 1.6 kV. The full scan range of mass spectrometry was 350–1600 m/z and the resolution was set to 60,000. The initial m/z of the secondary mass spectrometry was fixed at 100 and the resolution was set to 15,000. The ion fragmentation mode of high energy collisional dissociation (HCD) was set and the fragment ions were detected in Orbitrap, which was an ultrahigh field mass analyzer that could provide higher resolution and faster scanning speed. Databases including UniProt protein database, genomic annotation-based protein database, such as NCBI were selected for protein identification. The Mascot v2.3.02 software  was used to process the MS/MS data for analyzing the peptide sequences.https://research.bioinformatics.udel.edu/peptidematch/index.jsp). Peptide match service is one of the most popular tools on the PIR website that plays an important role in locating occurrences of a specific peptide [The protein names of the peptides were obtained from the Protein Information Resource (PIR) database  [The degree of hydrolysis (DH) was determined according to the method of Adler-Nissen  with som mmol/g) .w/w) sodium dodecyl sulfate, and 100 μL of β-mercaptoethanol. Distilled water was added to bring the total volume of the mixed solution up to 50 mL. Then the freshly prepared OPA solution was obtained. Subsequently, 100 μL of peptide solution was mixed with 2 mL of OPA solution. After the incubation for 2 min at room temperature, the absorbance of the reaction mixture was recorded spectrophotometrically at 340 nm by using a UV-visible spectrophotometer UV-3600Plus. Casein tryptone was used as a standard for quantification of peptide content. The peptide yield was determined by the ratio of peptide mass to protein mass.The protein content before simulated gastrointestinal digestion was analyzed by Kjeldahl method as described by Marcó et al. . The pep50) values, which meant the amount of inhibitor required to inactivate 50% of ACE activity under the experimental conditions were calculated from regression lines of a plot of % inhibition versus concentrations by using SPSS 13.0 software. All assays were performed in triplicate. ACE inhibitory activity was calculated by using the following equation:The ACE inhibitory activity was measured by using cary eclipse fluorescence spectrophotometer  according to a modified method of Li et al. . The samhttp://www.rcsb.org/pdb/home/home.do). To prepare the structure of ACE, all substrates, chloride ions, and water molecules were excluded in ACE model. Then the polar hydrogens were added to the ACE model. The number of poses per ligand was set to 20 to perform the molecular docking and the conformations for each peptide based on Total scores of Surflex-Dock were ranked [The molecular docking was carried out by using Surflex-Dock module that had an empirical scoring function and a patented searching engine ,56 in Sye ranked . The cone ranked . The intp < 0.05. All analyses were performed in triplicate. Data was expressed as the mean ± standard deviation and performed by one-way analysis of variation by using SPSS 13.0 for Windows  with Least Significant Difference (LSD) multiple range tests. The significant difference level was set at 50 value of (3.60 ± 0.10) μM among the peptides. Molecular docking results suggested that the ACE inhibition of GHIITVAR was mainly attributed to forming very strong hydrogen bonds with the S1 pocket (Ala354 and Tyr523), the S2 pocket  and the S1’ pocket (Glu162) after docking. It also bound to the active sites using a metal ion interaction (Zn701) and a hydrogen bond interaction with His383. Based on these results, sesame protein can be a promising ACE inhibitor in the prevention and treatment of hypertension. In addition, GHIITVAR can be used as a potential nutraceutical for development of functional foods. In conclusion, this study isolated and purified new ACE inhibitory peptides from sesame protein by using two stages of simulated gastrointestinal digestion in vitro. The DH, peptide yield, and ACE inhibition at different time points were determined during simulated gastrointestinal digestion, which demonstrated that the DH, peptide yield increased with the increase of digest time. The action of endogenous digestive enzymes increased the ACE inhibitory activity after simulated gastrointestinal digestion. SPDS was purified by ultrafiltration and SPDS-VII (<3 kDa) had strongest ACE inhibition. SPDS-VII was further purified by NGC Quest™ 10 Plus Chromatography System and finally 11 major peptides were identified by Nano UHPLC-ESI-MS/MS from peak 4. The peptide GHIITVAR derived from 11S globulin exhibited superior ACE inhibitory activity with the IC


This paper archives spatiotemporal volumetric moisture content (VMC) and associated precipitation datasets collected between 2012 and 2014 at different depths in two different New York City green infrastructure (GI) (e.g. bioretention) facilities, termed Site 1 and Site 2, respectively. The two sites are similar in both design and monitoring set up, and are located within two kilometers of one another, but differ in terms of hydraulic loading ratio (HLR). Both sites were designed and instrumented specifically to facilitate a comparison of the hydrologic fluxes within the two GI facilities. Site 1 receives only direct rainfall and is hydrologically isolated from the surrounding impervious surfaces (HLR = 1); Site 2 receives both direct precipitation and street runoff through a curb cut inlet (HLR = 3.8). Monitoring was conducted both inside (L plots) and outside (G plots) weighing lysimeters that were installed at both sites and planted with similar vegetation. Each L and G plot was equipped with five soil moisture sensors installed at 5, 10, 20, 30, and 50 cm depths in a circular pattern. This dataset is associated with the original research presented in “Observed Variability in Soil Moisture in Engineered Urban Green Infrastructure Systems and Linkages to Ecosystem Services  Urban stormwater utilities need performance data in order to justify and potentially expand/adapt this investment.13/m3 are shown in the figures. The accompanying *.csv files include all the raw data values .The dataset contains raw data collected at two different bioretention facilities . The raw2Soil moisture and precipitation data were collected in two bioretention facilities located within two kilometers of one another in Queens, New York City (NYC). The two NYC sites were profiled in 2018 as examples of nature-based solutions to stormwater in WWAP Site 1 a is a biThis paper archives data gathered from 2012 to 2014 using rain gauges and soil moisture sensors installed at both sites. Onsite precipitation was monitored with two, side-by-side Texas Electronics tipping bucket rain gages mounted at a height of four meters. The rain gages have a 0.2 m orifice and 0.254 mm resolution per tip. At each site, soil moisture monitoring was conducted at two locations, inside and outside weighing lysimeters, termed “L” and “G” plots, respectively. All locations were planted with similar vegetation. Each L and G plot was equipped with five soil moisture sensors installed at 5, 10, 20, 30, and 50 cm depths in a circular pattern to avoid electrical interference between them. The soil sensors were calibrated to the specific soil type. Data collected at each site was logged on a Campbell Scientific CR1000 data logger at 5-min time intervals and transmitted via a cell modem to a server for real time viewing.The lysimeter at Site 1 receives only direct precipitation, like the rest of the site. To ensure that the lysimeter at Site 2 is dosed with runoff at the same HLR as the rest of its site, a specially designed flow diversion box and orifice was installed at the inlet. The flow diversion box directs runoff to the lysimeter between mid-April and mid-October only. During the colder winter months, the Site 2 lysimeter receives direct rainfall only, to avoid pipe rupture due to water expansion during freezing conditions.The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.


The Getz region of West Antarctica is losing ice at an increasing rate; however, the forcing mechanisms remain unclear. Here we use satellite observations and an ice sheet model to measure the change in ice speed and mass balance of the drainage basin over the last 25-years. Our results show a mean increase in speed of 23.8 % between 1994 and 2018, with three glaciers accelerating by over 44 %. Speedup across the Getz basin is linear, with speedup and thinning directly correlated confirming the presence of dynamic imbalance. Since 1994, 315 Gt of ice has been lost contributing 0.9 ± 0.6 mm global mean sea level, with increased loss since 2010 caused by a snowfall reduction. Overall, dynamic imbalance accounts for two thirds of the mass loss from this region of West Antarctica over the past 25-years, with a longer-term response to ocean forcing the likely driving mechanism. The Getz region of West Antarctica is losing ice at an increasing rate; however, the forcing mechanisms remain unclear. Here we show for the first time that since 1994, widespread speedup has occurred on the majority of glaciers in the Getz drainage basin, with some glaciers speeding up by over 44 %. Observations have shown that ice loss from Antarctica is dominated by the low-lying, marine-based sectors of West Antarctica4, where glaciers in the Amundsen Sea Sector have thinned, accelerated, and grounding-lines have retreated since the 1940s8. Elsewhere, ice shelves on the Antarctic Peninsula have retreated9 and collapsed11, triggering a dynamic response as the flow of grounded ice is unbuttressed12. Dynamic imbalance in West Antarctica is driven by incursions of warm modified Circum-polar Deep Water (mCDW) melting the floating ice13, with the interannual and long-term variability of ocean temperatures linked to atmospheric forcing associated with the El Nino-Southern Oscillation (ENSO)15 and anthropogenic forcing16, respectively. The ice sheet contribution to the global sea level budget remains the greatest uncertainty in future projections of sea level rise17, driven in part by positive feedbacks such as the Marine Ice Sheet Instability (MISI)19, and with the most extreme scenarios (>1 m by 2100) only possible through the onset of Marine Ice Cliff Instability (MICI)21. Satellite data have shown that in Antarctica the dynamic ice loss ) is 86% greater than the modest reduction in surface mass (0.9 ± 1.1 mm sle) since the 1990s22. However, both long-term and emerging new dynamic signals must be accurately measured in order to better understand how ice sheets will change in the future. Since 2008, 88% of the observed ice speedup has occurred on glaciers located in the Amundsen Sea, Getz and Marguerite Bay sectors23. However, the timing and pace of dynamic imbalance is poorly characterised in regions that are observed less frequently, and uncertainty remains about the physical mechanisms driving this change.Over the last 25 years, the Antarctic Ice Sheet has contributed 7.6 ± 3.9 mm to global sea level2) of the West Antarctic Ice Sheet8. Ice flows from the ice sheet into the Getz Ice Shelf through 14 distinct glaciers that extend ~145 km inland, and flow at average speeds of over 500 m/year at the grounding line , the ice shelf is anchored at its seaward margin by eight large islands and over 23 pinning points which stabilise the ice shelf calving front, resulting in a relatively small area change over the past three decades26. Despite the absence of significant area change, strong ice shelf thinning (−16.1 m/decade) has been observed since the 1990s27, producing one of the largest sources of fresh water input to the Southern Ocean28, more than double that of the neighbouring Amundsen Sea ice shelves29. The complex network of topographic rises at the ice front channel ocean currents under the sub-ice shelf cavity, driving a highly localised spatial pattern of ice thinning, with the strongest rates observed at the grounding line30. Spatially variable ocean forcing is expected along the Getz coastline due to its zonal extent, about half the length of the West Antarctic Ice Sheet margin, and its location between the colder Ross and warmer Amundsen Seas25.The Getz drainage basin lies at the coastal margin of Marie Byrd Land, and covers 10.2% . The Flood, McCuddin and Executive Committee mountain ranges provide regions of high elevation bed topography at the ice divide, producing a broadly prograde bed slope across the basin32. This geometry prevents inland propagation of strong ice sheet thinning8  to the prevalent direction of atmospheric moisture flux33. While summer surface melt is limited to the lower elevation ice shelf33, interannual variability in Surface Mass Balance (SMB) is driven by the Amundsen Sea Low, which accounts 40% of the surface mass and 21% of the surface melt variability34. Partitioning the influence of both surface mass and ice dynamic signals in Antarctica is key to understanding the atmospheric and oceanic forcing mechanisms driving recent change35. Studies suggest that thinning of glaciers flowing into the Getz Ice Shelf are greater than the estimated increase in ice discharge alone, indicating that surface processes are at least in part responsible for the observed thinning36. However, a multi-decadal, continuous ice velocity record is required to perform a detailed assessment of the change in ice flux. In this study, we use satellite data to measure the change in ice speed of glaciers in the Getz drainage basin from 1994 to 2018, to assess the localised pattern of dynamic imbalance in this large and complex sector of West Antarctica. Our results show that between 1994 and 2018, widespread speedup has occurred on the majority of glaciers in the Getz drainage basin of West Antarctica. A mean speed increase of 23.8% was observed, with three glaciers accelerating by over 44%. Since 1994, 315 Gt of ice has been lost, contributing 0.9 ± 0.6 mm to global sea levels. Increased ice loss since 2010 was driven by a snowfall reduction, with dynamic imbalance likely driven by a longer-term response to ocean forcing.Over the past 30 years, the Getz drainage basin has lost ~410 Gt of ice massng8 Fig.  and makeOur velocity measurements show that the Getz coastline is characterised by 14 major flow units, with two glaciers reaching mean speeds of over 1 km/year in 2018, and regions of fast flow generally limited to within ~40 km of the grounding line Fig. . Nine of2) of the Getz drainage basin (Supplementary Table 2 (90%) and 155,518 km2 (90%) of the Getz basin, respectively  in the far West, where speeds have increased by 9.1 m/year2  Table , 3 and 4r2 Table . The smar2 Table  where ic94 Table . While wR2 = 0.74) as required by the theory of mass conservation37 . Our results extend and fill gaps in the spatial coverage, and show that the largest and most extensive ice speedup is concentrated on flow units 5 and 6 in the centre of the Getz drainage basin  have also undergone a significant increase in ice speed since 1994 (~25% on average), accounting for 39.7% of the total speedup in the region  correspond to a region of orographically driven high snowfall in excess of 3000 mm w.e./year, that is resolved by high spatial resolution (5.5 km) regional climate modelsean Fig. . Our mulean Fig. . The accean Fig.  and othe42, with periodic incursions of warm mCDW onto the continental shelf driving shorter term sub-decadal to decadal variability25. In the Getz region, annual mean temperature depth profiles collected from ship-based CTD sampling between 1994 and 2018, show annual variation in the depth of the thermocline dividing a cold and fresh upper layer from mCDW , where it can come into contact with and melt deeply grounded ice. Ocean circulation under Antarctic ice shelves is directed by the Coriolis force, which causes water to enter ice cavities on the Eastern side before being guided by the seabed and ice base geometry and exiting on the Western side. On the Getz Ice Shelf, Coriolis-driven circulation Fig.  brings i46 Fig. . The pat46 Fig. . We attr46 Fig. , in part00 m, whe45  at the grounding line on three glaciers ), and high speed up of over 20% on an additional three glaciers ) since 1994. The central region of the Getz drainage basin accounts for 46.8% of this acceleration, and contains the most spatially extensive areas of change. The pattern of ice speedup indicates a localised response on individual glaciers, demonstrating the value of high-resolution observations that resolve the detailed pattern of dynamic imbalance across the Getz drainage basin. On all glaciers, the speed increase coincides with regions of high surface lowering, with an ~50% speed up corresponding to an ~5% reduction in ice thickness. Our optimised model results show that 315 Gt of ice has been lost from the Getz drainage basin since 1994, contributing 0.9 ± 0.6 mm to global sea levels, and increasing the rate of ice loss by four times in the 2010s compared to the 1990s. The prograde bed topography makes the Getz region inherently less susceptible to unstable geometry driven feedbacks such as MISI or MICI compared with its Amundsen Sea neighbours, and indicates that long-term ocean warming may be driving the dynamic imbalance in this region of Antarctica. Consistent and temporally extensive sampling of both ocean temperatures and ice speed will help further our understanding of dynamic imbalance in remote areas of Antarctica in the future.55 and SAR interferometry techniques57. To map ice velocity using the Sentinel-1a and -1b satellites, we tracked the displacement of features near to or on the ice surface such as rifts, crevasses and stable amplitude variations in SAR images59. We apply the intensity feature tracking technique to temporally sequential pairs of Interferometric Wide (IW) swath mode Single Look Complex (SLC) SAR images60. The image pairs are co-registered using a bilinear polynomial function constrained by precise orbital state vectors, resulting in a coregistration accuracy of 5 cm60. The normalised cross correlation of SAR intensity features is calculated over evenly spaced image patches, with a window size of 64 (~0.9 km) by 256 (~0.6 km) pixels in the azimuth and range directions, to compute the 2D offset for each patch61. The results are converted into absolute displacement in ground range coordinates using the Antarctic digital elevation model (DEM) posted on a 1 km grid62. The final velocity measurements are mosaicked and averaged to produce an annual ice speed map for each year of the study period.We measured the ice speed of glaciers feeding the Getz Ice Shelf in West Antarctica over the last 25 years, using synthetic aperture radar (SAR) and optical satellite imagery acquired between January 1994 and December 2018  of the processed image size as sparse patchy data are an indicator of poor-quality measurements60. Errors in the velocity data are caused by imprecise co-registration of the SAR images, error in the auxiliary DEM, and atmospheric delay due to fluctuations in the ionosphere and tropospheric water vapour61. A spatially variable error measurement was computed for each velocity grid by multiplying the signal-to-noise ratio of the cross-correlation function with the ice speed60  filter over a 1 km by 1 km window, where values are rejected if the ice speed exceeds 30% of the mean value% 1.35 km of the p58 (Supplementary Table 63)  mode SLC SAR images acquired by the ERS-1 and ERS-2 satellites were used to measure ice speed in 1994, 1996 and 1998 using the intensity feature tracking technique65, calibrating the underlying model parameters—the vertically averaged viscosity and effective basal drag coefficients—and assuming that in the absence of observations they vary smoothly in both space and time . We investigated the impact of change in ice thickness on the ice discharge estimates, by reducing the ice thickness by 50 m at the flux gate. This is greater than the observed maximum thinning of 41.8 m over the full study period. We found that for 2018, this had a modest impact (1.5 %) on the total ice discharge. We assumed that the surface velocity in the direction normal to the flux gate equals the depth averaged speed with an assumed ice density of 917.2 kg/m3. We computed mass balance over the 25-year study period by subtracting the ice flux out of the basin from the time-varying annual mean SMB, using a 27-km regional climate model (RACMO 2.3p2)68. The error for the total mass balance estimate arises from the errors in the discharge and SMB. Discharge error is the combination of the percentage errors in ice thickness and velocity along the flux gate. For years of model discharge without a corresponding observation map, we assume the percentage error of the nearest observation year and add an additional standard deviation to the velocity error. The mass balance uncertainty is calculated as the discharge error combined with the error in SMB. We assume the uncertainty in the annual SMB trend to be 20% in line with previous studies69, and the cumulative uncertainty as the root sum square of the annual errors, assuming that they are not correlated over time. We calculated the SMB anomaly relative to the mean over the period 1979 to 2008, and accumulated over time68 (Fig. We calculated ice discharge from the Getz drainage basin using the 25-year record of optimised model ice flow (Supplementary Fig. e68 Fig. .Supplementary InfomationPeer Review File


Freezing has been widely recognized as the most common process for long-term preservation of perishable foods; however, unavoidable damages associated with ice crystal formation lead to unacceptable quality losses during storage. As an alternative, supercooling preservation has a great potential to extend the shelf-life and maintain quality attributes of fresh foods without freezing damage. Investigations for the application of external electric field (EF) and magnetic field (MF) have theorized that EF and MF appear to be able to control ice nucleation by interacting with water molecules in foods and biomaterials; however, many questions remain open in terms of their roles and influences on ice nucleation with little consensus in the literature and a lack of clear understanding of the underlying mechanisms. This review is focused on understanding of ice nucleation processes and introducing the applications of EF and MF for preservation of food and biological materials. The majority of foods are susceptible to spoilage due to mainly the growth of pathogenic bacteria and the enzymatic activities during storage. It is estimated that approximately 25–30% of perishable food is wasted because of the food spoilage and most of this wastage could be saved if stored properly  and magnetic field (MF) appear to influence the onset of ice crystal formation during supercooling since water consists of dipole molecules and is also a diamagnetic substance , the additional inclusion of water molecules gradually lowers the free energy and the ice embryo continues to grow, become thermodynamically favored, and towards crystallization  per unit volume of a supercooled liquid is expressed by an Arrhenius type relationship  in “the supercooled chamber” at the set temperature of − 3.5 ± 0.5 °C with UV irradiation treatment for 15 days. They showed that the supercooling preservation at − 3 °C effectively delayed the growth of microorganisms. Another point of view on supercooling was suggested from the same institute. Choi et al.  and the electric field intensity . The relationship between the electric flux density and the electric field intensity is represented as:−12 F/m, 2), B, Wb/m2 or Tesla) and the magnetic field intensity . The relationship between the magnetic field flux density and magnetic field intensity is defined as:7 H/m, Recently, several techniques have been proposed and developed to manipulate the nucleation processes; however, only EMFs have been employed in a commercialized refrigeration system  elevated the nucleation temperature of water. In contrast, Orlowska et al.  due to the induced electric dipole oscillation. Conversely, the physical mechanisms for AEF studies involving in direct contact with electrodes are more complex and include different effects such as the effect of an electric field, electric current, and air bubbles, etc. For example, pulsed AEF (1.78 × 102 V/m at 20 kHz) in direct contact with a sample was successfully applied to control the freezing behaviors of 0.9% NaCl solution, in particular for the formation of uniform and small ice crystals and reduction of the phase transition time   on ice nucleation and crystallization processes during freezing. However, unfortunately, experimental data in the literature have appeared to be contradictory and showed low reproducibility. Currently, the accurate mechanisms have not been completely elucidated, but the majority of studies suggest that hydrogen bonds between water molecules become strengthened under SMF and the molecules and/or water clusters are more ordered and stably distributed, therefore, ice nucleation is promoted (Cheng et al., According to the literature, SMF has been applied for not only simple matrices but only some complex biological materials. Lin et al.  tested tIn short, the main mechanism for OMF-assisted freezing in the literature is that OMF directly act upon water molecules by re-orientating and vibrating the molecules and/or break hydrogen bonds between water molecules, resulting in a large degree of supercooling and small ice crystal formation within biological materials such as animal tissues, foods, and living cells (Jha et al., Over the past few years, the beneficial effects of OMF on freezing of food and biomaterials have been claimed by a few investigators and manufacturers (Hirasawa et al., In conclusion, supercooling preservation has great potential to maintain food quality and extend the storage life of biological materials; however, the current state of the technology has not matured enough to be widely adopted in commercial applications. In spite of the fact that ice nucleation is one of the most important factors in conventional freezing processes and the supercooling preservation, it remains an uncontrolled variable due to the complexities of the phenomenon and the stochastic nature. The application of electric and magnetic fields has been shown the potential to control ice nucleation within foods and biomaterials. In summary, SEF and SMF are potentially able to induce ice nucleation in a supercooled product and AEF and OMF tend to exert the opposite effects. However, some of the mechanisms and detailed effects have not adequately elucidated yet and results in the literature appear contradictory, in particular, AEF and OMF’s impacts (summarized in Fig.


Further research identified α5 and α7 to be the receptors for NLS and the chromosome region maintenance 1 (CRM1) to be the receptor for the nuclear export signal. Moreover, we found that PRV VP11/12 interacts with EP0 and the stimulator of interferon genes protein (STING), whereas the NLS of VP11/12 is the important part for VP11/12 to interact with UL48. To our knowledge, this is the first study to provide reliable evidence verifying the nuclear localization of VP11/12 and its role as an additional shuttling tegument protein for PRV. In addition, this is also the first study to elucidate the interactions between PRV VP11/12 and EP0 as well as between PRV VP11/12 and STING, while identifying the precise interaction sites of PRV VP11/12 and VP16.Pseudorabies virus (PRV) is the etiological agent of Aujeszky's disease, which has caused severe economic loss in China since its re-emergence in 2011. UL46, a late gene of herpesvirus, codes for the abundant but non-essential viral phosphoproteins 11 and 12 (VP11/12). In this study, VP11/12 was found to localize inside both the nucleus and cytoplasm. The nuclear localization signal (NLS) of VP11/12 was identified as  Pseudorabies virus (PRV) is the pathogen for pseudorabies (PR), which is associated with nervous system disorders, respiratory disorders, and reproductive failure, resulting in massive economic loss to the swine industry in China, especially owing to the PRV variant that has emerged since 2011 .Varicellovirus in the subfamily Alphaherpesvirinae, which belongs to the family Herpesviridae. According to a previous study, all PRV genes contain homologs in at least one related alpha herpesvirus. Thus, most genes and certain protein names are derived from their locations within unique regions, in conformity with the prototypical herpes simplex virus type 1 (HSV-1)  . The str (HSV-1) , 5. The  (HSV-1) .The UL46 gene, a late gene of herpesvirus, encoding the abundant but non-essential viral phosphoproteins 11 and 12 (VP11/12) –9, is loHowever, UL46 is an important gene in herpesviruses and plays an important role in virion assembly and virus growth, especially in the secondary envelopment of viruses and in the inhibition of innate immunity upon viral infection. Moreover, VP11/12 has been reported to serve as the major component of virion tegument, in the form of conserved clusters and functional complexes with pUL47, pUL48, and pUL49 in HSV and PRV –15. AddiIn previous studies, subcellular localization of VP11/12 was observed in the cytoplasmic or perinuclear regions, in both plasmid-transfected cells and virus-infected cells. In our study, we investigated the intrinsic properties of VP11/12, in the absence of other viral proteins or in PRV JS-2012 infection, and found that VP11/12 localized to the nucleus, as observed by indirect immunofluorescence, and gradually appeared in the cytoplasm as well. Therefore, we sought to elucidate the specific mechanisms and vital functions of the nuclear localization signal (NLS) and nuclear export signal (NES) of VP11/12.Vero cells, HeLa cells, 293T cells, and PRV variant strain JS-2012 were obtained from the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences . Cos-7 cells and NIH/3T3 cells were kindly provided by the Shanghai Stem Cell Bank, Chinese Academy of Sciences . All cell lines were grown in Dulbecco's Modified Eagle Medium  supplemented with 10% fetal bovine serum  at 37°C.® HS DNA Polymerase was purchased from TaKaRa (Japan).Polyclonal antibody anti-UL46 was obtained from SHVRI-CAAS. Anti-FLAG and anti-HA monoclonal antibodies were purchased from Sigma-Aldrich (USA). DAPI and leptomycin B (LMB) were purchased from Beyotime Institute of Biotechnology . The monoclonal antibody anti-lamin B2 was purchased from Cell Signaling Technology . Donkey anti-mouse IgG secondary antibodies, Alexa Fluor 594 and Alexa Fluor 488, were purchased from Invitrogen . Anti-GFP monoclonal antibodies, HRP-conjugated goat anti-mouse IgG, and HRP-conjugated goat anti-rabbit IgG were purchased from ProteinTech (USA). All restriction endonucleases used for cloning procedures were purchased from New England Biolabs , and homologous recombinases were procured from Vazyme Biotech Co. Ltd. . PrimeStarKP257591) genomic DNA, using primers that incorporated EcoRI sites and homologous sequences in plasmids, p3xFlag-CMV, pEGFP-C3, or pCAGGS. The UL46 fragments were inserted into vectors, p3xFlag-CMV, pEGFP-C3, and pCAGGS, individually, to generate UL46-FLAG, UL46-EGFP, and UL46-HA, respectively. For the positive control, full-length UL47 of PRV JS-2012 genomic DNA was cloned into the EcoRI site of p3xFlag-CMV to generate UL47-FLAG. The vector pEGFP-C3 served as the negative control because it could express the enhanced green fluorescent protein (EGFP) protein.The full-length UL46 open reading frame, including both the start codon methionine and the stop codon, was PCR-amplified from PRV strain JS-2012  according to the manufacturer's instructions. Samples were then digested with DNase I to remove trace amounts of contaminating DNA. The integrity of total RNA was checked for cDNA synthesis using SuperScript™ III reverse transcriptase .in vitro, a monolayer of Vero cells, HeLa cells, NIH/3T3 cells, or Cos-7 cells were plated overnight on coverslips in six-well plates  in DMEM  containing 10% FBS, at a density of 2 × 105 cells per well, before transfection. Cells were transfected with mixtures of 1.5–2.0 μg plasmid DNA, Opti-MEM , and FuGENE HD Transfection Reagent , according to the manufacturer's instructions. Indirect immunofluorescence assay (IFA) was then performed. For certain samples, LMB was added into the culture medium at a final concentration of 10 and 20 ng/ml for 4 h.To express all proteins After transfection for 24 h, cells were fixed in ice-cold absolute methanol at −20°C for 30 min, washed three times in PBS, and blocked with PBS containing 5% bull serum albumin  for 1 h at 28°C. Subsequently, cells were washed three times with PBS and incubated with anti-Flag monoclonal antibody as the primary antibody in six-well plates for 1 h at 37°C. After washing with PBS three times, donkey anti-rabbit IgG secondary antibody, Alexa Fluor 594  in PBS, was added to plates for 1 h at 37°C. After each incubation step, cells were washed three times extensively with PBS and stained with DAPI (Beyotime Biotechnology). Samples were analyzed using a Zeiss LSM 880 laser scanning confocal microscope. Images were processed with Adobe Photoshop.These experiments were carried out as described previously (31); 293T cells were transfected alone or in combination with plasmids UL46-HA, α1-FLAG, α3-FLAG, α4-FLAG, and α6-FLAG using Lipofectamine 3000 . At 24-h post transfection (hpt), the cells were washed twice with cold PBS and lysed with IP lysis buffer (Thermo Fisher) mixed with inhibitors for protease and phosphatases (BioTool). Except for the inputs of lysates, other cell lysates were incubated with anti-HA agarose  at 4°C overnight. The immunoprecipitates were washed four times with IP lysis buffer and then subjected to western blotting analysis.P < 0.05.Data are presented as mean ± SD. Statistical analysis was performed using GraphPad Prism version 7.0 . Differences were considered statistically significant at 2IRRARGTRRASWKDASRRVTEGRTRASC29 and 662PRPRTRADDGLYQQPRPVIDLTGHRASRRKSW693  and in the C-terminus of FLAG in the p3xFlag-CMV vector (UL46-FLAG). The plasmid DNA of UL46-EGFP was transfected into live Vero cells and HeLa cells; 24 h later, cells were fixed with cold methanol. Alternatively, the plasmid of the UL46-FLAG was transfected into NIH/3T3 cells. After 24 h, they were fixed with cold methanol and washed three times with PBS. They were incubated with anti-flag monoclonal antibodies for 1 h at 37°C and then washed three times with PBS. The cells were then incubated with anti-mouse IgG secondary antibody Alexa Fluor 594 for 1 h at 37°C. In order to differentiate between nuclei and cytoplasm, all cells were stained with DAPI and assessed by a Zeiss LSM 880 laser scanning confocal microscope. Consequently, as shown in A previous study revealed LMB is an inhibitor of CRM1, one of the major nuclear export receptors for leucine-rich NES-containing proteins . We analVP11/12 has been reported to be localized in the cytoplasm of HSV-1 and HSV-2. Michael Murphy et al.  hypothesAll the above results demonstrate that VP11/12 translocates into the nucleus from the cytoplasm and is transported into the cytoplasm from the nucleus whether viral proteins were present or not. Moreover, CRM1 was identified as the nuclear export receptor for VP11/12.Because of UL46 protein, approximately 100 kDa, UL46-EGFP was unable to enter the nucleus without the assistance of additional proteins , 27, unlTo identify the NLS, the N-terminal 48-residue peptide was truncated, and the following related plasmids were generated: UL46(1–40)-EGFP, UL46(1–30)-EGFP, UL46(1–25)-EGFP, and UL46(1–20)-EGFP . IFA revMoreover, to confirm whether VP11/12 contains other NLSs, a plasmid with deletion of the N-terminal 1–20 amino acids, UL46(21–696), was constructed and transfected into Vero cells in six-well plates. UL46(21–696) was not localized in the nucleus but rather was distributed in the cytoplasm . Further3RRARGTRRASWKDASR18 peptide was considered to have sufficient residues to guide VP11/12 to the nucleus. To further ensure the effectiveness of the UL46(3–18), we constructed the plasmid NLS-UL35-FLAG fusing with the NLS and PRV UL35 and UL35-FLAG. PRV VP26 encoded by gene UL35 was verified only by cytoplasmic localization without other PRV viral proteins  were cloned into EGFP-C3, thereby constructing a series of deletions of UL46 . Subcellproteins , 30. TheNuclear localization signals consist of many basic amino acids, especially arginine . There aThe results described above indicate that VP11/12 contains a classical NLS, which may direct it to the nucleus. Thus, we considered the importin α/β pathway as a mediator of VP11/12 nuclear import. Seven different isoforms of importin have been characterized in mammalian cells , 33. To To verify that the NLS of VP11/12 is the interaction site between VP11/12 and α5 or α7, we constructed a plasmid, ΔNLS-UL46, in which the NLS of VP11/12 was truncated and contained the vector HA-tagged PCAGGS. The ΔNLS-UL46 was co-immunoprecipitated with α5- or α7-FLAG by HA-tagged beads. The results showed that α5 and α7 were detected in the input samples but not in the IP samples. These data revealed that α5 or α7 was possibly required for guiding VP11/12 to the nucleus and that deletion of NLS from VP11/12 abolished the VP11/12 binding to α5- or α7-FLAG. This further indicated that the α5 and α7 might meditate the nuclear import of VP11/12 .UL48, encoding VP16, plays vital roles in promoting the assembly of a multicomponent complex with Oct-1 and HCF-1, to induce the expression of viral immediate–early genes  and secoEP0, the homolog of ICP0 in HSV-1, is a transactivating protein that can enhance the infectivity of PRV genomic DNA, couples with IE180 –45, and HSV-1 VP11/12 has been reported to interact with STING via its 100 N-terminal amino acids . TherefoHSV-1 VP11/12 is a multifunctional tegument protein that interacts with many viral proteins and cellular proteins in immune signaling pathways, thereby contributing to several biological processes , 47–49. Nucleocytoplasmic shuttling proteins contain NLSs and NESs. Herein, we demonstrated that PRV VP11/12 shuttles between the nucleus and cytoplasm, which was the first report on nucleocytoplasmic shuttling of herpesvirus VP11/12. We also found that VP11/12 was to possibly localize in the nucleus and performed related experiments to identify the NLS. Furthermore, we confirmed that the nuclear export of VP11/12 occurred through its interaction with CRM1, because 20 ng/ml of LMB inhibited its nuclear export.3RRARGTRRASWKDASR18 was identified as the functional NLS. VP11/12 appeared to have been phosphorylated heavily in at least 23 sites when HSV-1 infected, and phosphorylation played important roles in modulating VP11/12 abundance with respect to ICP0 localization and interactions -EGFP. Subsequent experiments demonstrated that UL46(1–48)-EGFP could be seen in both the nucleus and the cytoplasm . TherefoIn a previous study, HSV-1 VP11/12 had been proven to interact with the host protein STING  and the The PRV VP11/12 interacted with PRV pUL48, and more importantly, the NLS of VP11/12 interacted with pUL48/VP16, the key activator of lytic infection by initiating the lytic program . A previ3RRARGTRRASWKDASR18. Furthermore, we identified interactions between PRV VP11/12 and EP0, PRV VP11/12 and pUL48, and PRV VP11/12 and the host protein, STING, which is critical for the growth of PRV. The significance of these interactions requires further investigation.In summary, we verified the nucleocytoplasmic shuttling of VP11/12 and identified its NLS, The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.JX: experiment, writing, and design. FG: supervise and revise. JW: experiment and design. HZhe: supervise and design. XC, YL, HZhu, and XF: experiment. WT, YJ, LL, and NK: supervise. GL and GT: funding. All authors contributed to the article and approved the submitted version.The 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.