Mast cells are tissue-resident immune cells that function in both innate and adaptive immunity through the release of both preformed granule-stored mediators, and newly generated proinflammatory mediators that contribute to the generation of both the early and late phases of the allergic inflammatory response. Although mast cells can be activated by a vast array of mediators to contribute to homeostasis and pathophysiology in diverse settings and contexts, in this review, we will focus on the canonical setting of IgE-mediated activation and allergic inflammation. IgE-dependent activation of mast cells occurs through the high affinity IgE receptor, FcεRI, which is a multimeric receptor complex that, once crosslinked by antigen, triggers a cascade of signaling to generate a robust response in mast cells. Here, we discuss FcεRI structure and function, and describe established and emerging roles of the β subunit of FcεRI (FcεRIβ) in regulating mast cell function and FcεRI trafficking and signaling. We discuss current approaches to target IgE and FcεRI signaling and emerging approaches that could target FcεRIβ specifically. We examine how alternative splicing of FcεRIβ alters protein function and how manipulation of splicing could be employed as a therapeutic approach. Targeting FcεRI directly and/or IgE binding to FcεRI are promising approaches to therapeutics for allergic inflammation. The characteristic role of FcεRIβ in both trafficking and signaling of the FcεRI receptor complex, the specificity to IgE-mediated activation pathways, and the preferential expression in mast cells and basophils, makes FcεRIβ an excellent, but challenging, candidate for therapeutic strategies in allergy and asthma, if targeting can be realized. Initially, they circulate in the blood as committed progenitors, and are then recruited into tissues where they mature and terminally differentiate into mast cells . Mast cview see ).aK = 1010–1011 M−1 that is orders of magnitude above that of IgG binding to any of the FcRs [onk ≈ 105 M−1 s−1 for IgE binding with FcεRI being comparable with IgG associating with its high affinity receptor, FcγRI, the dissociation rate for IgE (offk ≈ 10−5 s−1) is at least an order of magnitude slower than that of IgG [Tissue-resident mast cells express the high affinity IgE receptor, FcεRI, on their surface. FcεRI is a multichain immunorecognition receptor (MIRR) that binds to monovalent IgE with very high affinity the FcRs . Despitet of IgG ,7,8 and t of IgG . Howevert of IgG . Therefot of IgG . This prt of IgG ,14. Signt of IgG ).The canonical composition of FcεRI is that of a tetrameric receptor complex with a total of seven transmembrane regions within the complex . The tet2 or tetrameric αβγ2 complex, and whether the receptor complex is constitutively expressed or inducible. Mast cells and basophils are known to constitutively express FcεRI and express the tetrameric αβγ2 form of the receptor. The presence of FcεRIβ and the function of the FcεRI complex is less clear for immune cells outside of the mast cell and basophil compartments. Dendritic cells, neutrophils, eosinophils, monocytes and other cells can be induced to express FcεRI that are either in the tetrameric αβγ2, or trimeric αγ2 configurations [Mast cells are not the only cell type that express FcεRI. In humans, the receptor is also expressed by basophils, dendritic cells, eosinophils, monocytes, neutrophils, platelets and smooth muscle cells ,22,23,24urations ,22,23,24urations ). Howeve2 and αβγ2 complexes are capable of binding IgE with high affinity, the differential expression of tetrameric αβγ2 and trimeric αγ2 FcεRI on distinct immune cell populations suggests that the two configurations of the receptor have divergent roles in immunity. In mast cells, FcεRI-mediated cell activation and the distinct roles of each subunit of the tetramer have been well characterized [Although both αγcterized   from intracellular stores via the generation of the intracellular secondary messenger inositol triphosphate (IP3). Depletion of these stores leads to activation of the calcium-release activated ion channel Orai1 within the plasma membrane, which opens to allow Ca2+ influx [2+ fluxes, Orai1 activity triggers a variety of calcium-dependent events, including gene expression , exocytiewed in ,46,47,48iewed in ).In comparison to the tetrameric isoform, the properties and roles of trimeric FcεRI are more obscure—a consequence, in part, of differences between humans and mice. Human FcεRIα possesses an extracellular domain that facilitates trafficking with the γ-subunits to the cell surface in the absence of the β-subunit , whereasAs key effector cells in allergy and inflammation, mast cells and basophils are ideal therapeutic targets. Additionally, due to the high affinity binding of IgE to FcεRI, thereby allowing the cells to be primed for activation for months , inhibitDespite these significant benefits, omalizumab, for a variety of known and unknown reasons, can have variable efficacies in different allergic disease settings and their patient subsets , . When co-aggregated with FcεRI, phosphorylation of this ITIM leads to recruitment of SH2 domain-containing inositol 5-phosphatase (SHIP). SHIP recruitment inhibits FcεRI activation and calcium influx ,97 by prInhibiting intracellular signals, such as tyrosine kinase inhibitors targeting SYK or modulating Raf kinase inhibitor protein (RKIP) activity, represent other potential options for allergic therapeutics, due to their roles in IgE-mediated mast cell activation ,105,106.MS4A2, have been linked to allergy and asthma susceptibility [MS4A2 polymorphisms with allergy and asthma is not consistent [MS4A2 cDNA containing mutations associated with asthma has previously failed to alter FcεRIβ function [MS4A2, which are linked to asthma susceptibility, remain elusive [The above discussion demonstrates that efforts to therapeutically suppress FcεRI-mediated mast cell activation are ongoing and a need remains for alternative approaches. To this end, FcεRIβ presents a potentially appealing target. In addition to its role in amplifying IgE-mediated mast cell activation, polymorphisms in the gene encoding FcεRIβ, tibility ,114,115,nsistent ,117,118 function ,120. The elusive ,122,123.MS4A2 with functional outcomes in disease were unsuccessful, the subsequent elucidation of alternative splicing of FcεRIβ mRNA and the functional differences of alternate isoforms have established new avenues of study and reignited interest in the therapeutic potential of FcεRIβ. Alternative splicing enables a single gene to generate a variety of different mRNA transcripts and protein isoforms and is thereby an important regulatory component of eukaryotic gene expression [Although initial attempts to associate polymorphisms in pression . Splicinpression , recogni2 receptor complex from the endoplasmic reticulum . In pariewed in ,119,127.iewed in . Since tMS4A2 (MS4A2 variant 2-FcεRIβT) caused by inclusion of intron 5 results in a premature in-frame stop codon and loss of the downstream third and fourth transmembrane regions, as well as the C terminal ITAM [T can associate with FcεRIα. However, FcεRIβT competes with full-length FcεRIβ for FcεRIα binding, and redirects FcεRIα to endosomes and proteasomal degradation rather than to the plasma membrane [In human basophils and cord blood-derived mast cells, a truncated splice variant of nal ITAM . Since tnal ITAM , FcεRIβTmembrane . Consequmembrane .MS4A2 variant 3, which excludes exon 3 and produces a truncated isoform (t-FcεRIβ) that lacks the first two transmembrane regions [MS4A2 variant 3 also plays a role in IgE-mediated mast cell activation. Following mast cell activation, t-FcεRIβ interacts with calmodulin via a putative calmodulin-binding domain and, most likely through its C-terminal ITAM, also binds Fyn kinase, GRB2-associated-binding protein (Gab)-2, and the phosphoinositide 3 kinase (PI3K) p85 subunit [Human mast cells also express  regions . The fir regions , and thu regions . Adenovi regions . However subunit . Togethe subunit ,131.MS4A2 splice variants in mast cells, it becomes apparent that alternative splicing of FcεRIβ pre-mRNA selectively removes domains in the protein that are critical for specific functions. Therefore, in addition to regulation of FcεRI expression by transcription of the FcεRI subunits, alternative splicing of FcεRIβ pre-mRNA may regulate FcεRI trafficking to the cell surface, since alternative splicing dictates the intracellular trafficking and function of FcεRIβ. Thus, altered splicing of MS4A2 could have implications in susceptibility to allergic diseases. Moreover, manipulation of MS4A2 splicing to favor a particular phenotype, such as aberrant FcεRIα trafficking, could have therapeutic potential.By determining the various roles of MS4A2, splice switching oligonucleotides (SSOs) targeting exon 3 of MS4A2 (herein referred to as FcεRIβ SSOs) have been employed by our group [To manipulate splicing of ur group . SSOs arur group ). These ur group ,135,136)ur group .SSOs can promote the inclusion or removal of exons from mature mRNA. In the case of the latter, SSOs induce skipping of a specific exon in mature mRNA by binding to splicing sites in precursor-mRNA, resulting in a steric block of the spliceosome machinery proteins from binding to the site . Recent MS4A2, which prevents FcεRIβ from binding to and trafficking FcεRIα (To this end, FcεRIβ SSOs have been used to induce exon skipping and force the cell to preferentially produce the alternatively spliced t-FcεRIβ isoform . Specifig FcεRIα . In mousThe loss of surface FcεRIα should impact IgE-mediated mast cell activation because, even in the presence of high levels of IgE, mast cells lacking surface FcεRIα will not have the capacity to bind IgE. Indeed, in BMMCs and, to a lesser extent, LAD2 cells, FcεRIβ SSO-induced loss of surface FcεRIα corresponds with inhibition of IgE-mediated degranulation  Figure . ImportaMS4A2 gene.Targeting FcεRI and IgE-mediated mast cell and basophil activation has great therapeutic potential for allergic diseases. In addition to the established approaches that target IgE and binding of IgE to FcεRI, targeting FcεRI trafficking and signaling may also prove effective in IgE-mediated diseases. Identifying genes and proteins that play important roles in the FcεRI pathway is a critical step to finding novel targets for therapeutics. However, alternative splicing is often overlooked and splice variants can not only provide information for how pathways are regulated; once they are understood, the alternative splicing can also be utilized as a way to target those pathways. By altering the splicing of non-mutated but pathologically associated genes, the therapeutic potential of SSOs extends beyond diseases driven by genetic mutations. For allergic diseases and asthma, FcεRIβ is an ideal target for SSO therapy, since it has a characteristic role in IgE-mediated mast cell activation that is inhibited by SSO-mediated alternative splicing of the


The significance of long-term teleconnections derived from the anomalous climatic conditions of El Niño has been a highly debated topic, where the remote response of coastal hydrodynamics and marine ecosystems to El Niño conditions is not completely understood. The 14-year long data from a ship-borne acoustic Doppler current profiler was used to examine the El Niño’s impact, in particular, 2009 and 2015 El Niño events, on oceanic and biological processes in coastal regions across the Korea/Tsushima Strait. Here, it was revealed that the summer volume transport could be decreased by 8.7% (from 2.46 ± 0.39 to 2.24 ± 0.26 Sv) due to the anomalous northerly winds in the developing year of El Niño. Furthermore, the fall mean volume backscattering strength could be decreased by 1.8% (from − 97.09 ± 2.14 to − 98.84 ± 2.10 dB) due to the decreased surface solar radiation after the El Niño events. Overall, 2009 and 2015 El Niño events remotely affected volume transport and zooplankton abundance across the Korea/Tsushima Strait through climatic teleconnections. In particular, the El Niño-Southern Oscillation (ENSO) is the most dominant climate signal that affects the global climate with anomalies in the sea surface temperature (SST) and wind system3. Previous observations indicated that the offshore SST and climate signals across East Asia are closely related to these El Niño patterns despite the long distance from the Central Pacific5. Hence, the climate is generally warmer and wetter in East Asia during an El Niño winter and subsequent spring. Because climate variability in the open ocean affects regional areas, their associated teleconnection has been highlighted to predict the interannual variability of regional marine ecosystems. Ref.6 showed that the timing of the vertical migration and the abundance of zooplankton under seasonally varying sea ice correlated with the ENSO in the Amundsen Sea, Antarctica. A close relationship was observed between the El Niño seasons and East Asian regions  biomass, where differences in the SST anomaly (SSTA) and precipitation patterns lead to changes in the biomass location following the latitudinal migration of the salinity front7.Climate change has major effects on atmospheric and oceanic conditions, leading to changes in precipitation, wind, and ocean currents10. For example, the coastal hydrodynamic models included complex interactions between Pacific basin-scale wind forcing and local wind stress in order to predict the seasonal cycle of volume transport, but did not include interannual climate variabilities11. Ref.10 suggested that the typhoon-dependent nutrient supply largely regulates the abundance and composition of the phytoplankton assemblages. These studies were generally based on short-term (< 2 years) observations rather than long-term monitoring efforts10. Therefore, volume transport and zooplankton abundance potentially depend on the poorly understood interplay between oceanic processes and climate systems, and require an in-depth understanding of the impact of El Niño on the local weather and marine ecosystems. This study examined the effects of two El Niño events, which occurred in 2009 and 2015, on the seasonal and interannual variability in volume transport and mean volume backscattering strength (MVBS) from a ship-borne acoustic Doppler current profiler (ADCP) across the Korea/Tsushima Strait  as a secondary circulation5. This leads to an anomalous cyclonic circulation in the western North Pacific via atmospheric teleconnections with northerly winds  decreased by a reduction in surface solar radiation (SSR)  Fig. . Low cloSR) Fig. . These a6 because the detectable particle size exceeds the size of plankton19. Despite this potential complication, the spatial and temporal variability in zooplankton abundance can be predicted from the magnitude of phytoplankton blooms20. The positive relationship between MVBS and the chlorophyll-a concentration indicates the capability of MVBS to detect zooplankton  with increasing (decreasing) SST , El Niño research focuses mostly on longer time series (> 30 years). To alleviate this concern, the climatic impacts on the ocean are compared in terms of the superimposed impacts of two El Niño events, instead of dividing them into the effects of separate EP/CP-El Niño. La Niña events are also a potential factor for determining hydrodynamic and biological processes across the KTS. Because the impacts of EP- and CP-El Niño on volume transport tend to be stronger in amplitude, and exhibit more pronounced spatial diversities3, La Niña events were excluded from this study. The arguments are based mainly on a series of statistical analyses.The temporal extent of the study period explains the potential limitations of the statistical significance of these results. Because El Niño commonly occurs on longer timescales (Eastern Pacific El Niño (EP-El Niño): 3–7 years; Central Pacific El Niño (CP-El Niño): 2–3 years, 10–15 years3 showed that EP-El Niño is generally associated with basin-scale equatorial winds and large eastward shifts of the tropical Pacific convection zone, as well as the strong discharge of the heat content from the equatorial band. In contrast, CP-El Niño is associated more commonly with local wind feedback, weak shifts of the convection zone, earlier termination, and only limited discharge of the ocean heat content. This supports the present findings that volume transport and MVBS were lowest when the NINO3 was the highest (2015 El Niño) during the entire analyzed period. On the other hand, Ref.26 further argued that the response of ENSO over East Asia is strong when the ENSO-Pacific Decadal Oscillation (PDO) relationship is “in phase”, i.e., either El Niño and a positive PDO phase or La Niña and a negative PDO phase, compared to that when the ENSO-PDO is “out of phase”. This is because the atmospheric response over the western tropical Pacific is enhanced when the ENSO and PDO are in phase in which the SSTA has the same sign in the central-to-eastern tropical Pacific. In fact, the impact of El Niño on volume transport and MVBS could be enhanced, when NINO3 and PDO are in phase  on the East Asian oceanic variances compared to CP-El Niño . Ref.The impacts of low-latitude climate systems on the interannual variability including ENSO events on local volume transport and zooplankton is not completely understood. This study found that El Niño caused an 8.7% (from 2.46 ± 0.39 to 2.24 ± 0.26 Sv) decrease in volume transport and a 1.8% (from − 97.09 ± 2.14 to − 98.84 ± 2.10 dB) decrease in MVBS due to anomalous northerly winds and anomalously increased cloud coverage, respectively, indicating that there may exist a linkage between El Niño and oceanic responses through atmospheric teleconnection. Therefore, the hydrodynamics and marine ecosystems across the KTS could be sensitive to low-latitude climate signals including ENSO events. This suggests that the changes in physical background in the tropical Pacific will have major impacts on the weather and marine ecosystems through variations in volume transport and zooplankton abundance within the East Asian regions. In particular, oceanic responses were significant when NINO3 and PDO were in phase, so that SSTA has the same sign in the central-to-eastern tropical Pacific. Our results further underscore the relative importance of the influence of EP-El Niño on the East Asian oceanic variances compared to CP-El Niño. Overall, the changes in volume transport and MVBS are a good proxy for the teleconnection between El Niño and the mid-latitude oceanic processes.27. The Kuroshio Current flows northward along the continental shelf break, where mixing processes between the East China Sea and the Kuroshio Current occur28. The Kuroshio Current consists mainly of three water masses  with different origins and nutrient properties; hence, the characteristics of each part of the Kuroshio Current depend on the mixing rate28. The Kuroshio Current detaches from the continental shelf break at 30° N and 128–129° E, where it meets cold, fresh continental shelf water and forms a density front and frontal meanders. Subsequent mixing with continental shelf water then forms the origin of the Tsushima Warm Current27.The Tsushima Warm Current is a branch of the Kuroshio Current that originates east of the Philippine coast by the bifurcation of the North Equatorial Current10. The Tsushima Warm Current is divided by the Tsushima Islands into the western and eastern channels. The western channel has a narrow and hollow shape with a width of 40 km and a depth of 150–200 m, while the eastern channel is relatively wide and shallow, reaching widths and depths of 140 km and 110 m, respectively30. Volume transport through the western channel flows northeast along the east coast of the Korean Peninsula, while a deep southwestern countercurrent introduces cold water to the bottom layers27.The KTS is a complex channel system with a width and length of 180 km and 330 km, respectively  and four additional tidal constituents  as well as annual  collected by New Camellia in 2016 was compared with MVBS during the same period using linear regression. Furthermore, New Camellia MVBS was compared with the MVBS calculated from the bottom-moored ADCP data, which was collected between August 8th and 17th, 2014 by an up-looking 300-kHz ADCP mounted on a trawl-resistant bottom mount .The acoustic backscatter strength was extracted for each depth bin from the ADCP to calculate MVBS by applying a simplified sonar Eq.ion mg l− collectehttps://www.esrl.noaa.gov/). NINO3 indicates the mean-removed and 3-month moving averaged SSTA over the 5° N–5° S and 150–90° W region, which indicates the EP-El Niño events. El Niño (La Niña) events are identified by NINO3 exceeding (being less than) 0.5 °C (− 0.5 °C) for more than 8 months34. In order to compare NINO indices with climate and ADCP data, composite analyses and 3-month moving average were then applied.The ADCP data were compared with NINO and PDO indices from 2004 to 2017 to account for the spatial variability in atmospheric teleconnection throughout the tropical Pacific region (The climate data was provided as an ERA5 dataset by the European Centre for Medium-Range Weather Forecasts (ECMWF). The dataset included the following: the SST, sea level pressure, 10 m-above zonal and horizontal wind speeds, precipitation, high cloud cover, medium cloud cover, low cloud cover, total cloud cover, surface thermal radiation, and SSR. Each dataset was normalized to the standard deviation because the MVBS, volume transport, and climate data largely depend on the time of the year. Composite analysis was then applied to describe the yearly anomalies without the average of each calendar month. The 3-month moving averaged composite values were then averaged and divided into four groups based on the season. In this paper, winter is defined as December of the previous year, January and February; spring is defined as March, April, and May; summer is defined as June, July, and August; fall is defined as September, October, and November. Note that all the indices were compared based on their anomalous state. The climate data was extracted between 33.5–35.5° N and 127.75–130.25° E to compare to the NINO indices. Supplementary Table Supplementary Information.


Bacterial ice nucleators(INs) are among the most effective icenucleators known and are relevant for freezing processes in agriculture,the atmosphere, and the biosphere. Their ability to facilitate iceformation is due to specialized ice-nucleating proteins (INPs) anchoredto the outer bacterial cell membrane, enabling the crystallizationof water at temperatures up to −2 °C. In this Perspective,we highlight the importance of functional aggregation of INPs forthe exceptionally high ice nucleation activity of bacterial ice nucleators.We emphasize that the bacterial cell membrane, as well as environmentalconditions, is crucial for a precise functional INP aggregation. Interdisciplinaryapproaches combining high-throughput droplet freezing assays withadvanced physicochemical tools and protein biochemistry are neededto link changes in protein structure or protein–water interactionswith changes on the functional level. NumerousINs have been identified and their ice nucleation efficiencies aretypically characterized using droplet freezing assays.9 In such assays, a large number of droplets containing a well-definedconcentration of INs is gradually cooled down and the fraction offrozen droplets as a function of temperature is recorded. The temperatureat which half of the droplets are frozen, T50, provides a direct measure for the efficacy of the IN. While mineraldust-based INs  play amajor role in the atmosphere owing to their ubiquity, the ice nucleationefficiency of biological INs derived from bacteria, fungi, lichen,or plants is much higher.5 Despite itssignificance and the acceleration of research in this field in recentyears, several questions on the molecular-level mechanisms of heterogeneousice nucleation remain unanswered. This makes it difficult to predictthe decisive properties of efficient INs and their role in the environment.Understanding such molecular-level mechanisms could point to novelways of triggering ice nucleation, desirable not only for artificialsnow, for instance, but also for new artificial anti-icing surfaces.12Freezingprocesses in the atmosphere have a significant influenceon the formation of clouds, on precipitation patterns, and on Earth’senergy balance.Pseudomonas in the 1970s.14 Subsequently, several other ice-nucleating bacteria belonging tospecies in the Pseudomonadaceae, Enterobacteriaceae, Xanthomonadaceae, and Lysinibacillus families havebeen identified.17 The best-characterized bacterial INs are Pseudomonas syringae, which enable ice nucleationat temperatures at −2 °C. The ability of bacteria to facilitateice formation is attributed to specialized proteins anchored to theouter bacterial cell membrane. As a plant pathogen, P. syringae causes frost injury to the plant tissueby increasing the nucleation temperature of water, which enables accessto nutrients.9 Moreover, like many otherice-nucleating microbes, P. syringae was identified in ice, hail, and snow, indicating that they mightcontribute to freezing processes in the atmosphere.18 Theunique standing of P. syringae as asource of exceptional bacterial INs is further emphasized by its commercializationas Snomax. This artificial snowmaking product consists of extractsof sterilized P. syringae.Ice-nucleation activity in bacteria was first discovered in 25Thebiomolecules responsible for bacterial ice nucleation are largeice nucleation proteins (INPs) anchored to the outer membranes ofthe bacterial cells, as schematically shown in P. syringae has beendeduced and is widely used to model its structure as shownin 28 The INP consists of three domains: (1) a central repeating domain(CRD) comprising ∼81% of the total sequence, (2) an N-terminaldomain comprising ∼15% of the sequence, and (3) a C-terminalunique domain (∼4%). The CRD has been proposed to contain theice nucleation site of the INPs, and molecular simulations have shownthat the active site consists of similarly effective hydrophobic TxTand hydrophilic ExSxT amino acid motifs.29The amino acid sequenceof the INPs of 30 A β-helical motif has also been used to modelthe structure of bacterial INPs,31 on thebasis of the idea that AFPs and INPs share similar folds and ice-bindingmotifs.32The large size and embedment into the membrane still hamperexperimentalattempts to solve the three-dimensional structure and associated molecular-leveldetails of the INPs. In contrast, the structures of antifreeze proteins(AFPs) containing similar TxT motifs have been solved, oftentimesrevealing β-solenoid folds.T50 but a wider range ofnucleation temperatures. On the basis of extensive freezing assaysof P. syringae for different concentrations,three distinct classes of INs have been proposed.34A centralenigma of bacterial ice nucleation arises from the broaddistribution of threshold nucleation temperatures ranging from −2to −12 °C. This is reflected in freezing assays that shownot one single 35 Southworth et al. revealed a nonlinear relationshipbetween ice nucleation activity and the concentration of INPs in bacterialcells.36 Together, those findings indicatethat the different activation temperatures can be explained by aggregationof INPs, thereby varying the accumulated size of the ice nucleationsite. These protein aggregates provide another example of how proteinaggregation can have beneficial effects to cellular systems.37 Simulations have addressed the role of sizeand aggregation of the proteins on the freezing temperature and providedquantitative predictions of the ice nucleation temperature vs thenumber of proteins in the aggregates, as well as to the distance betweenthe monomers in the aggregates.38 On thebasis of freezing assays, the predominant and least efficient fractionof bacterial INs active at ∼−7 °C, ClassC, has been attributed to small aggregates of INPs (5–10INPs38).33 Themost active Class A INs are active at temperaturesup to ∼−2 °C and consist of the largest aggregatesof the INPs (>30 INPs38).33 Class B INs are rarely observed and responsiblefor freezingbetween ∼−5 and ∼−7 °C. Aggregationof the INPs in the cell membrane was described in several studiesand it has further been suggested that the membrane plays a majorrole in enabling the highly active Class A INs.40Govindarajan and Lindow showed that the largest structuresof INsreach the highest threshold temperature, i.e., nucleate ice most efficiently.41 Observations at the functional level can beaccompanied by molecular-scale investigations using spectroscopictools. Circular dichroism and infrared spectroscopy provide informationon the secondary structure, while surface-specific vibrational sum-frequencygeneration spectroscopy (SFG) is a powerful tool to investigate themolecular-level details of the interface of bacterial INPs and water.46 The biophysical and spectroscopic investigations are further highlydependent on sample quality. Recent progress in ice-affinity purificationmethods now allows for isolating ice-binding proteins directly fromnatural sources and with high purity.49 In the studies presented here, we utilized inactivated extractsfrom P. syringae, commercially availableunder the product name Snomax (Snomax Int.).Progress in unraveling themechanism underlying bacterial ice nucleationrequires advanced physicochemical methods and interdisciplinary approaches.Essential for any investigation of INs are droplet freezing assays.High-throughput assays, like the Twin-plate Ice Nucleation Assay (TINA),now enable the simultaneous measurement of complete dilution series with robust statistics, enablingthe cumulative representation of the complete range of present INs.P. syringae under different environmental conditions. All cumulative freezingspectra are composed of measurements of a 10-fold dilution series.The fraction of frozen droplets (fice)measurements shown in P. syringae INs in pure water (gray curves) in Nm) is calculated using Vali’s equation50 and represents the number of ice nucleatorsper unit weight that are active above a certain temperature. The twostrong increases at ∼−3 and ∼−7.5 °Ccorrespond to the large aggregates (Class A INs) and the smaller aggregates(Class C INs), respectively. The two increases are followed by plateaus,which indicate that fewer INs are active in those temperature ranges.512O). The freezing spectrum is shifted∼+4 °C, which is consistent with the expected shift of+3.82 °C based on the higher melting point of D2O.Turner et al. previously described a third intermediate Class B ofINs, active at around −5 °C, and that examining the effectsof substituting D2O for H2O allows for differentiationof the different classes on the basis of their isotope-induced shiftsin nucleation threshold.33 As apparentfrom P. syringae in H2O andD2O do occur. Measurements in D2O show a largernumber of Class A INs and fewer Class C INs. We explain the observeddifferences with higher rigidities of INPs in D2O and fewerstructural fluctuations of the INP aggregates due to the strongerintramolecular D-bonds.5234P. syringae as a function of pH.42 Upon lowering the solution pH, the first rise at ∼−3°C (Class A) gradually decreases and shifts to lower temperatureswhile the fraction of INs active at ∼−7.5 °C (ClassC) increases. There seems to be a clear interconversion of Class Aspecies into Class C species with decreasing pH. At a pH of ∼4.5,we observe that only Class C INs remain active.Severalstudies have reported that pH changes of the aqueous solutionor the addition of cosolutes affects the Class A INs differently thanClass C.42 In SFG spectroscopy,a broadband infrared (IR) beam is used to probe the molecular vibrationsin a given frequency region  in D2O at room temperature and 1 °C above the melting point and showedthat the SFG signal in the O–D stretch region is increasedand red-shifted at low temperature, indicating an increase in thealignment of the water molecules.43 ShortenedINPs with low ice nucleation activity expressed in E. coli showed a similar effect, and the observationwas attributed to an activation of INPs at lower temperature and theability to order water, which increases close to the respective freezingtemperature.45 While providing much neededexperimental insights into the INP/water interface, these studiesand interpretations must be taken with a caveat, given that more recentlyit has been shown that water ordering at lower temperatures observedwith SFG . The important role of functional aggregation is furtherunderlined by simulation studies, which have shown that not only ClassA but also the smaller Class C INs active at around −7.5 °Care a product of functional aggregation of the proteins and mergingof their active sites.38 Our studies ofpurified INPs from P. syringae haveunderlined the importance of the membrane for the formation of ClassA aggregates,44 emphasizing its essentialrole for the ice nucleation activity. We hypothesize that the formationof Class C aggregates might have another molecular mechanism thanthe membrane-associated mechanism responsible for forming the largerClass A aggregates. Clarification of whether the membrane’srole lies merely in providing a matrix or whether it is part of theactive ice nucleation site is another critical step for unravelingthe molecular origin of bacterial ice nucleation.58 In addition to unsolved questions regarding the 3D structureof the INP monomer and the interfacial structure of water at the functionalsite of the INP, information on the precise numbers of INPs in theaggregates, their alignment, and which interactions  drive the aggregation is needed  close to water’smelting temperature. The process of bacterial ice nucleation at warmtemperatures requires an appropriate pH value and intact INP structures.s needed 4.Understanding the molecular-level processes driving bacterial icenucleation may provide further insights into the role of biologicalINs in the environment. Answering these questions will likely alsoenable the community to unravel how nature precisely aligns INPs tobe the most efficient ice nucleators known and illuminate how thisstrategy can be copied for new freezing products and technologies.


Prior studies show that glycogen synthase kinase 3β (GSK3β) contributes to cardiac ischemic injury and cardiac hypertrophy. GSK3β is constitutionally active and phosphorylation of GSK3β at serine 9 (S9) inactivates the kinase and promotes cellular growth. GSK3β is also phosphorylated at serine 389 (S389), but the significance of this phosphorylation in the heart is not known. We analyzed GSK3β S389 phosphorylation in diseased hearts and utilized overexpression of GSK3β carrying ser→ala mutations at S9 (S9A) and S389 (S389A) to study the biological function of constitutively active GSK3β in primary cardiomyocytes. We found that phosphorylation of GSK3β at S389 was increased in left ventricular samples from patients with dilated cardiomyopathy and ischemic cardiomyopathy, and in hearts of mice subjected to thoracic aortic constriction. Overexpression of either GSK3β S9A or S389A reduced the viability of cardiomyocytes subjected to hypoxia–reoxygenation. Overexpression of double GSK3β mutant (S9A/S389A) further reduced cardiomyocyte viability. Determination of protein synthesis showed that overexpression of GSK3β S389A or GSK3β S9A/S389A increased both basal and agonist-induced cardiomyocyte growth. Mechanistically, GSK3β S389A mutation was associated with activation of mTOR complex 1 signaling. In conclusion, our data suggest that phosphorylation of GSK3β at S389 enhances cardiomyocyte survival and protects from cardiomyocyte hypertrophy. Cardiovascular diseases are the leading cause of morbidity and mortality in the world ,2. New tGSK3β is active in unstimulated cells, where it phosphorylates and thus inhibits growth stimulating substrates. There are more than 40 substrates for GSK3 and an additional 500 other potential candidates have been identified ,9. GSK3 The key upstream regulator of GSK3β is PI3K-Akt pathway, which is activated by multiple growth factors that couple their cell surface receptors to specific PI3K isoforms. Activated Akt phosphorylates GSK3β at S9 residue, which inactivates the kinase and allows cardiomyocyte hypertrophic growth to occur . In addiThe aim of this study was to investigate the significance of phosphorylation of GSK3β at S389 in the myocardium. We found that phosphorylation of GSK3β at S389 was increased in diseased human and mouse hearts. Studies in primary rat cardiomyocytes showed that GSK3β S389 phosphorylation enhances cardiomyocyte survival and protects from cardiomyocyte hypertrophy. Analysis for potential downstream mechanisms indicates that GSK3β S389 regulates the activation of mTORC1 signaling.We first investigated if phosphorylation of GSK3β is dysregulated in left ventricular samples of explanted hearts from patients with end-stage dilated or ischemic cardiomyopathy. In comparison, GSK3β phosphorylation was analyzed from left ventricular samples of accidental death victims with no history of cardiovascular disease or evidence of cardiovascular disease at autopsy. We found that phosphorylation of GSK3β at S389 was increased both in dilated and ischemic cardiomyopathy hearts A. In addWe then analyzed if phosphorylation of GSK3β is altered in experimental heart failure model in mice. Wild type mice were subjected to TAC for 6 weeks and left ventricular tissue samples were collected for analysis. Western blot analysis showed that phosphorylation of S389 in GSK3β was increased in response to chronic hemodynamic pressure overload by TAC B. On theAs phosphorylation of GSK3β at S389 was augmented in diseased hearts in both human and mice, we wanted to examine the biological role of GSK3β phosphorylation in cardiomyocytes more closely. For this, we utilized adenoviral overexpression of GSK3β with mutations on key phosphorylation sites of the kinase. Neonatal rat ventricular cardiomyocytes were transduced with adenoviruses encoding for wildtype GSK3β (WT), GSK3β with serine 9 to alanine mutation (S9A), GSK3β with serine 389 to alanine mutation (S389A), and double mutated GSK3β with S9A and S389A mutations (S9A/S389A). LacZ was used as a control virus.We first assessed for the possible regulation of GSK3β S9 phosphorylation by GSK3β S389. Analysis for GSK3β phosphorylation shows that phenylephrine (PE) only induces weak phosphorylation of GSK3β at S9 and S389 in cardiomyocytes transduced with LacZ A–C. CellCells overexpressing GSK3β S389A show abolished basal and PE-induced GSK3β S389 phosphorylation and, interestingly, also reduction in the basal and PE-induced GSK3β S9 phosphorylation B. On theTo study the significance of GSK3β S389 phosphorylation in regulating cardiomyocyte viability, we transduced adult rat cardiomyocytes with WT GSK3β or GSK3β variants and analyzed cell death by measuring the release of adenylate kinase from necrotic cardiomyocytes . OverexpJC1 assay was performed to investigate the effect of GSK3β on mitochondrial membrane potential. Overexpression of WT GSK3β or GSK3β variants had no effect on mitochondrial membrane potential in cardiomyocytes at normoxia B. In carp38 MAPK is activated in response to cardiac ischemia and previous data suggest that inhibition of p38 during ischemia/reperfusion has a cardioprotective effect ,25,26. T3H]-leucine incorporation showed that overexpression of GSK3β S389A or GSK3β S9A/S389A induced both basal and FGF-induced protein synthesis in neonatal cardiomyocytes  is the mechanism of cardioprotection afforded by GSK3β inhibition . Data frGSK3β phosphorylation site at S389 is phosphorylated by p38 MAPK and this also inactivates GSK3β . Data byThe role of GSK3β S9 in cardiac hypertrophy and remodeling has been studied in different in vivo models, including myocardial infarction and TAC. In GSK3α/β-S21A-S9A double knock-in mouse model, where GSK3 is supposed to be constitutively active, preventing hypertrophy, there was no difference in hypertrophic response and remodeling between WT or transgenic mice after permanent MI . Antos eIn cell culture models, hypertrophic agonists such as endothelin-1 increase the phosphorylation of GSK3β Ser9 and thus inhibit GSK3β at least partly, leading to hypertrophic response . In the Active GSK3 phosphorylates its substrate β-catenin and thus triggers the β-catenin destabilization ,41. InhiGSK3β has several targets that play a role in regulation of cardiomyocyte growth. Nuclear GSK3β phosphorylates and inhibits multiple proteins, including transcription factors such as GATA-4, members of the NF-AT family, and c-Myc ,43,44. AmTOR is a central regulator of cellular energy homeostasis and cell growth . Early sp70S6K is a central downstream effector of mTORC1  and p70SInterestingly, analysis of both human and mouse samples show regulation of GSK3β phosphorylation in the diseased myocardium. Analysis of cardiac samples from patients with end-stage ICM or DCM showed an increase in GSK3β phosphorylation at S9 and S389, and left ventricle samples of mice subjected to TAC showed an increase in GSK3β S389 phosphorylation. Data from our studies with isolated cardiomyocytes suggest that the noted increase in GSK3β S389 phosphorylation may counteract the development of cardiac hypertrophy in the diseased myocardium.In summary, our data suggest that phosphorylation of GSK3β at serine 389 attenuates hypertrophic cardiomyocyte growth and offers protection from cardiomyocyte death during hypoxic stress. Mechanistically, defective GSK3β S389 phosphorylation is associated with increased phosphorylation of mTORC1 target kinase p70S6K. These data provide novel evidence for the role of GSK3β Ser389 in regulating GSK3β function in cardiomyocytes.The left ventricular samples from patients with ischemic and dilated cardiomyopathy were obtained from patients undergoing cardiac transplantation in Helsinki University Hospital between 2014 and 2019. The investigation conformed to the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Helsinki and Uusimaa Hospital District, Finland . A written informed consent was obtained from the patients. The control samples were collected as part of the FinGesture study, which has systematically collected both clinical and autopsy data from sudden cardiac death victims between 1998 and 2018 in Northern Finland. Control samples were from victims of traffic accidents with no history or evidence of cardiovascular diseases at autopsy. The study complies with the Declaration of Helsinki and was approved by the Ethics Committee of the Northern Ostrobothnia Hospital District, Finland and the National Supervisory Authority for Welfare and Health, Finland . Permits to use data from medico-legal death investigations were obtained from the Finnish Institute for Health and Welfare and the Regional State Administrative Agency of Northern Finland.To induce hemodynamic pressure overload, eight-week-old male C57BL/6 mice were subjected for thoracic aortic constriction (TAC) as described before . The aorThe experimental protocol was approved by the National Animal Experiment Board . The investigation was carried out in accordance with the national regulations of the usage and welfare of laboratory animals and conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.We used adenoviral vectors for gene delivery and expression of GSK3β as well as its mutants in cultured cardiomyocytes. We used three GSK3β viruses with mutations in phosphorylation sites from serine to alanine: ser9ala (S9A), ser389ala (S389A), and double mutation ser9ala/ser389ala (S9A/S389A). LacZ and wild-type GSK3β were used as control.g, at +4 ℃, 24 h). Viruses were diluted in phosphate buffered saline (PBS) and stored at −70 ℃. Titer of viruses was determined by Ad Easy Viral Titer Kit . As a control virus, adenovirus containing E. coli β-galactosidase coding LacZ gene was used.Adenoviruses were generated as described before  with mod2 in PBS. Cells were fractionated by repeated incubations in collagenase at +37 ℃. After incubations, the cell suspension was centrifuged twice , and the supernatant was discarded and replaced with fresh Dulbecco’s Modified Eagle’s medium/F-12 , including 10% fetal bovine serum , 2.56 mM L-glutamine , and penicillin–streptomycin 100 IU/mL (Sigma-Aldrich). Isolated cells were preplated for 2 h to remove fibroblasts. Then remnant myocytes were replated at a density of 1.8–2 × 105/cm2 and incubated overnight in DMEM/F-12 supplemented with 10% FBS. After that, the medium was changed to complete serum-free culture medium , 1 μM insulin + 32 nM selenium + 2.8 mM sodium pyruvate + 5.64 μg/mL transferrin , 1 nM 3′-3′-5′triiodotyronine (Sigma-Aldrich), 2.56 mM l-glutamine, 100 IU/mL penicillin–streptomycin). After experiments, medium samples were collected and wells were rinsed twice with cold PBS and quickly frozen at −70 °C.Neonatal rat ventricular cardiomyocytes (NRVMs) were prepared from 2–4 day-old Sprague–Dawley rats, as described earlier . Briefly2 and the heart was excised rapidly. The heart was cannulated through aorta and retrogradily perfused with Hepes-buffered Tyrode’s solution supplemented with 1 mg/mL collagenase type II  and 2,3 -butandione-monoxime  until digested. Ventricular tissue was homogenized, and myocytes were collected with low-speed centrifugation. After Ca2+ reintroduction, cardiomyocytes were resuspended and cultivated in αMEM supplemented with Earle’s salt  containing 5% FBS , 20 mM HEPES (Sigma), insulin-transferrin-selenium (Gibco), 10 mM BDM, 2 mM l-glutamine and penicillin–streptomycin in laminin-coated  plates at a density of 1–1.5 × 104/cm2. After the cells had attached, medium was changed, and FBS was replaced with 0.01% BSA when starting the experiment. After experiments, medium samples were collected, and wells were rinsed twice with PBS and quickly frozen at −70 °C.Adult rat ventricular cardiomyocytes (ARVMs) were isolated from 8–12 week-old male Sprague–Dawley-rats as described before  and modiPenicillin-streptomycin was left out of the medium where viruses were added to cells. In NRVM cultures, adenoviruses were added at 3 MOI 24(-48) h after plating, depending on the designed experiment. In ARVM cultures, adenoviruses were added at 100 MOI 2 h after plating.3H]-leucine incorporation into the cells. NRVM or ARVM were cultured in 24-well plates, and when hypertrophic stimulus was added, medium was supplemented with [3H]-leucine (5 µCi/mL). After 24 h cells were lysed and processed for measurement of incorporated [3H]-leucine  by liquid scintillation counter .Cardiomyocyte protein synthesis was measured by analyzing a radioactively labeled [2 with ProOx C21 O2/CO2 controller  for 4 h and after were allowed to reoxygenate for 3 h. Medium samples for cell viability assay were collected after reoxygenation.ARVM cells were infected with viruses 2 h after plating. After 24 h, medium was changed to plainer version without FBS, penicillin–streptomycin, and HEPES. The cells were incubated in hypoxic C-Chamber with oxygen levels controlled at 0.1% in 5% COAnalysis of necrotic cell death was performed by measuring the release of adenylate kinase from ruptured ARVM into the cell culture medium by using a bioluminescent ToxiLight bioassay  according to manufacturer’s instructions.v/v) Triton-X100, 2.5 mM sodium pyrophosphate, 1 mM β -glycerophosphate, and 1 mM Na3VO4 (pH 7.5) supplemented with 1 mM dithiothreitol , protease inhibitors (1:100), and phosphatase inhibitors . Samples were then centrifuged at 10,000× g for 5 min at 4 °C and the supernatant was collected. Protein concentrations were determined by the Bradford method. Protein extracts were matched for protein concentration and stored denatured in SDS loading buffer at −70 °C. Nuclear and cytosolic proteins were extracted as described [Frozen cells, scraped from cell culture plates and frozen cardiac tissue samples that were ground in liquid nitrogen, were dissolved and homogenized in ice-cold lysis buffer containing 20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1%  for 30 min at +37 °C. Cells were washed once with cell culture medium and hypoxia experiment was performed as described above. After hypoxia, fluorescent readings for JC-1 aggregate emission (590 nm) and JC-1 monomer emission (530 nm) were measured with Varioskan Flash (Thermo Scientific) using Skanlt Software for Varioskan Flash version 2.4.5 (Thermo Scientific).t-test or Mann–Whitney U-test was performed. Data are shown as mean ± SD. Differences were considered statistically significant at the level of p < 0.05.Statistical analysis was performed with IBM SPSS Statistics software. To compare multiple groups, one-way ANOVA was used, followed by Tukey post hoc test for equal variances, or the Games–Howell post hoc test for unequal variances. The Kruskall–Wallis test was performed when data did not represent normal distribution. Normality of variables was tested with Kolmogorov–Smirnov and Shapiro–Wilk tests. When two groups were compared, Student’s


N-glycopeptides, mainly located in the Hp glycosylation site Asn207. Four N-glycopeptides were found to have significant changes in abundance between cirrhosis and HCC samples (p < 0.05). Strategic combinations of the significant N-glycopeptides, either with alpha-fetoprotein (AFP) or themselves, better estimate the areas under the curve (AUC) of their respective receiver operating characteristic (ROC) curves with respect to AFP. The combination of AFP with the isomeric sialylated fucosylated N-glycopeptides Asn207 + 5-6-1-2 and Asn207 + 5-6-1-3, resulted with an AUC value of 0.98, while the AUC value for AFP alone was 0.85. When comparing cirrhosis vs. early HCC, the isomeric N-glycopeptide Asn207 + 5-6-0-1 better estimated AUC with respect to AFP .Currently, surveillance strategies have inadequate performance for cirrhosis and early detection of hepatocellular carcinoma (HCC). The glycosylation of serum haptoglobin has shown to have significant differences between cirrhosis and HCC, thus can be used for diagnosis. We performed a comprehensive liquid chromatography—parallel reaction monitoring—mass spectrometry (LC-PRM-MS) approach, where a targeted parallel reaction monitoring (PRM) strategy was coupled to a powerful LC system, to study the site-specific isomerism of haptoglobin (Hp) extracted from cirrhosis and HCC patients. We found that our strategy was able to identify a large number of isomeric  Glycosylation alterations of serum proteins are commonly associated with the development of several cancer types; therefore, changes in protein glycosylation may play an important role in disease diagnosis ,2,3. TheN-glycopeptides have described the heterogeneity of their glycosylation sites, and have identified potential sialylated fucosylated N-glycopeptide structures as potential biomolecules to differentiate between both diseases [N-glycopeptide structures, could increase the possibility of finding haptoglobin site-specific N-glycopeptides that can differentiate between cirrhosis and the early stages of HCC. Moreover, the glycosylation site information, obtained by glycoproteomic analysis, would be an important factor in understanding how aberrant glycosylation is related to disease progression [In recent years, glycomic studies of serum haptoglobin have identified important isomeric structures, most of them being sialylated fucosylated glycans, with significant differences in abundance between patients with cirrhosis and HCC ,3. Analydiseases ,11,12. Agression .N-glycopeptide structures, the MS sensitivity needs to be enhanced and the LC separation improved. The current strategies to increase glycopeptide sensitivity use either enrichment or fractionation methods [The structural identification and quantitation of the isomeric site-specific glycosylation of haptoglobin remains challenging, due to the low abundance of important sialylated fucosylated structures ,2,10,14. methods ,16,17. E methods ,19,20,21n identification of the scanned ions, which is an important drawback in the evaluation of complex biological samples [n spectra of the targeted ions, where the analytes can be accurately identified based on their m/z ratio and fragmentation patterns, and quantified using their most abundant and constant fragments [An MS-based approach that can provide enhanced sensitivity is multiple reaction monitoring (MRM), which increases the glycopeptide detection by scanning the specific ions that represent the target structures. MRM increases the analyte detection sensitivity, but is limited by its inability to perform MS samples ,23. Alteragments ,22. For ragments . In contragments ,17,25.N-glycopeptides. The second strategy was based on a MS-PRM approach, directed to enhance the detection sensitivity of important low-abundance sialylated and fucosylated glycopeptides that change in abundance during the development of HCC [N-glycopeptide isomeric structures. Of significance, the statistical analysis of the data found seven haptoglobin N-glycopeptides that can be used alone, combined with AFP, or combined themselves, to better differentiate between cirrhosis and HCC group samples than previous approaches.In this study, we integrated two strategies to evaluate the microheterogeneity of serum haptoglobin, focusing on the glycosylation sites Asn184, Asn207, and Asn241. Haptoglobin was extracted from serum samples of patients with NASH cirrhosis and NASH-related HCC. The first strategy involved the use of a long nano-C18 column (50 cm), to ensure a wide separation of the tryptic and Glu-C digested haptoglobin isomeric t of HCC ,26. We wN-glycopeptides of serum haptoglobin, to characterize the alterations in site-specific glycopeptide forms between cirrhosis and HCC. The experimental workflow is shown in A targeted PRM approach was applied for the quantitation of the intact N-glycopeptides from the glycosylation site Asn211, in a reproducible number of samples, to apply reliable evaluation [N-glycopeptides present in the samples, based on monoisotopic mass, charge, retention time, and MS2 spectra , which can be observed after a two-step enzymatic digestion, using trypsin and Glu-C . In thisaluation ,12. A po spectra .N-glycopeptide structure was targeted in the LC-MS-PRM approach on the Orbitrap QExactive (Thermo). The selections of the precursor ions for all the identified glycopeptide structures was based on the signal intensity, so that in each case, the most abundant ion was used in the PRM strategy. Three charged precursor ions were the common ionic glycopeptide species in the three evaluated glycosylation sites, except for the glycopeptides with tri- and tetra-antennary and mono-, di-, and tri-sialylated glycans attached to the glycosylation site Asn241; where these structures presented four charged ions  software . The peptide moiety was described as MVSHHN184LTTGATLINE = Asn184, NLFLN207HSE = Asn207, and VVLHPN241YSQVDIGLIK = Asn241.The specific information for each detected ged ions . The eneon sites . Six of software . After tobserved . For alle number . The cor5Hex6Neu5Ac3 as an example. m/z value of 1181.7977, it eluted in a retention time of about 72 min, and it produced monocharged fragment ions, principally of the core glycan portion . 4Hex5FucNeuAc, HexNAc5Hex6FucNeuAc3, and HexNAc5Hex6Fuc2NeuAc3. All of these structures showed significant changes in abundance between the two disease states [N-glycopeptides was addressed, based on the use of a long C18 column (50 cm) and high temperature. The strategy was adopted from the work of Ji et al., where they reported the separation of isomeric O- and N-glycopeptides from tryptic-digested α1-acid glycoprotein (AGP), and evaluated the effects of temperature on the isomeric separation as well [Based on our early research efforts, as described by Huang et al. and Zhu et al., the isomeric glycan profile of serum haptoglobin, and its glycosylation site heterogeneity, were determined ,10. These states . The dev as well .N-glycopeptide forms from the tryptic/Glu-C-digested haptoglobin, as shown in N-glycopeptides in the glycosylation sites were as follows: 13 isomeric structures that corresponded to 11 N-glycopeptide forms in the glycosylation site Asn184; 44 isomeric structures that corresponded to 19 N-glycopeptide forms in the glycosylation site Asn207; and 16 isomeric structures that corresponded to 12 N-glycopeptide forms in the glycosylation site Asn241. The glycosylation site Asn207 was found to have the most abundant diversity of N-glycopeptides, as well as the most abundant isomeric N-glycopeptide structures.We were able to accurately identify and quantify a total of 73 isomeric structures that corresponded to 42 4Hex6Neu5Ac was only observed in the glycosylation site Asn184; the structure HexNAc3Hex4Neu5Ac was only observed in the site Asn207; and the structure HexNAc6 Hex7Neu5Ac3 was observed only in the site Asn241, which matched with the findings of Zhu et al. [4Hex5Neu5Ac2 (4-5-0-2) was present in the three sites, but, interestingly, two isomeric glycans were observed only in the site Asn207  software, using the normalized data for each glycosylation site  was performed, to evaluate the ability of the obtained data to differentiate between cirrhosis and HCC. The analysis was performed with the MarkerViewion site . The PCAion site a and VVLion site c showed ion site b. The clion site . Additioion site , where tion site , and in ion site .Site-specific heat maps were obtained for each of the following glycosylation sites: Asn184, Asn207, and Asn241 , and use241YSQVDIGLIK showed changes in abundance for the glycan structures 4-5-0-1 and 4-5-0-2; these glycans were more abundant in the cirrhosis than the HCC samples, The isomeric separation that was achieved by our analytical strategy increased the number of glycopeptide structures in the glycosylation site NLFLN207HSE, and thus its structural information (microheterogeneity), 241YSQVDIGLIK, did not show significant changes in abundance for sialylated and sialylated fucosylated glycans, The site-specific glycome changes associated with group-glycan types was also evaluated, using pie graphs. For comparison, the data were separated into the following three main glycan groups: sialylated, sialylated fucosylated, and other structures . As expeN-glycopeptides resolved by our LC-PRM-MS strategy, upgrade the differentiation between the evaluated sample groups. Most of the changes were observed in the glycosylation site NLFLN207HSE, which is information that elucidated the position of the protein glycosylation differences between the cirrhosis and HCC samples.The results observed in the PCA plots, heat maps, and pie graphs showed consistent glycosylation differences in serum haptoglobin between the cirrhosis and HCC samples. In addition, the important number of isomeric N-glycopeptides between cirrhosis and HCC, we further focused on investigating the differentially relative abundances of N-glycopeptides between both diseases. Initially, seventeen site-specific N-glycopeptides were found to have the most significant differences in abundance between cirrhosis and HCC, N-glycopeptide significance was evaluated by adjusting both the sample cohorts to the same gender ratio. Three sample sets were tested; N-glycopeptides. The large number of parameters used in this evaluation can increase the probability that the observed results were accidental. Therefore, “Bonferroni correction” was applied to the observed p-values. The corrected p-values can be observed in N-glycopeptides were found to have significant differences in abundance between cirrhosis and HCC, despite the gender factor. All of them were found in the glycosylation site NLFLN207HSE. The significant N-glycopeptides observed were as follows: the tri-antennary sialylated isomeric glycopeptides Asn207 + 5-6-0-1 isomer 2 and the Asn207 + 5-6-0-2 isomer 1; and the sialylated fucosylated isomeric glycopeptides Asn207 + 5-6-1-3 isomers 1 and 2. The statistical comparation of these N-glycopeptides between gender groups showed that no significant bias was introduced by gender, p-values, and cartoons of the N-glycopeptides with the most significant changes between the cirrhosis and HCC samples. Among these structures, the isomeric N-glycopeptide Asn207 + 5-6-1-3 showed the best capabilities differentiating between both the sample groups, with a p-value of 0.001 for the isomeric form one and a p-value of 0.005 for the isomeric form two, and when the glycopeptide was processed as a single structure, the p-value was 0.001. Furthermore, we previously reported the significance of the glycan structure “5-6-1-3” in our haptoglobin glycomics studies of HCC [p-values for other N-glycopeptides with significant changes were 0.001 and 0.038 for the glycopeptides Asn207 + 5-6-0-1 isomer 2 and Asn207 + 5-6-0-2 isomer 1, respectively. Our findings strongly correlated with the reported glycosylation alterations of serum haptoglobin in hepatic cancer. Ang et al. reported the increase in glycan fucosylation, the decrease in sialylated glycans, and the increase in α2,6 sialic acid linkage [N-glycopeptides and the significant changes described between cirrhosis and HCC by our analytical strategy, x fucosylation [N-glycopeptides Asn207 + 5-6-1-3 and Asn207 + 6-7-1-1, N-glycopeptides with significant changes, Since our LC-PRM-MS approach provided clear differences in site-specific  linkage , these csylation , which iN-glycopeptides to differentiate between cirrhosis and early HCC, the relative abundance of the glycopeptides was compared between the cirrhosis and HCC TNM 1 samples  values of their corresponding ROC curves to describe the cirrhosis and HCC relative abundance differences  is a graphical plot that illustrates the diagnostic ability of a binary classifier system, such as two related diseases or two stages in the progression of a particular disease. Thus, we evaluate the performance of the isomeric ferences . Initial of 0.88 .N-glycopeptide analyses have the advantage that different structures observed in a single assay can be combined, to gain sensitivity and specificity in the differentiation of two sets of samples. Therefore, we evaluated different glycopeptides as complement of AFP and different groups of glycopeptides that showed common changes between the cirrhosis and HCC samples  is another HCC biomarker that was also evaluated in the same cohorts, and the AUC values were lower than the values observed using the GALAD score, and lower than those observed in our strategy .Although the investigation presented here was based on a small sample number in the comparative cohort. The principal outcome of our LC-PRM-MS approach intended to elucidate a strategy that is capable of robustly identifying site-specific changes in glycoprotein-related diseases. Under this strategy, we were able to observe that several changes in protein glycosylation happen at the micro level, such as the glycan isomerism. Additionally, the analytical strategy used provided a high certainty in the identification of relatively small biomolecules present in complex biological samples. Precise and accurate identification can be achieved using the fingerprint mass spectra of the target molecules, which is an important advantage, due the lack of standards in bioanalysis. Regardless of the sample size, a proof of the consistent results is the strong correlation of our data with previous studies. Moreover, we incorporated additional supporting parameters that increased AFP sensitivity and specificity, even in the early HCC stages. As was mentioned earlier, the application of a PRM approach requires expensive and highly accurate LC-MS systems, which is not necessarily a disadvantage, because the discovery of biomarker molecules that are capable to use in disease diagnosis is of high importance for human health. Besides, after the discovery process is concluded, the analysis of single or groups of biomolecules can be easily transferred to a common and accessible analytical technique, such as HPLC.Trypsin/Lys-C mix mass spectrometry grade and Glu-C sequencing grade were purchased from Promega . The anti-human haptoglobin antibody was purchased from Abcam . Acetonitrile, methanol, and mass spectrometry-grade water were purchased from Fisher Scientific . Serum samples were provided by the University of Michigan and the University of Texas Southwestern Hospital according to IRB approval; 15 cases of liver NASH cirrhosis, and 15 cases of NASH-related HCC. The clinical information associated with the samples used in this study is summarized in Haptoglobin was purified from a 20 μL aliquot of serum for each patient using an HPLC-based antibody-immobilized method developed in house as previously reported ,10. Befo4HCO3 and denatured at 90 °C in a water bath for 15 min. The denatured glycoprotein was reduced by the addition of 0.5 μL of 200 mM dithiothreitol (DTT) and incubation at 60 °C for 45 min. Then, the glycoprotein was alkylated by the addition of 2.0 μL of 200 mM iodoacetamide (IAA) and incubation at 37 °C for 45 min. A second addition of 0.5 μL of 200 mM DTT and incubation at 37 °C for 30 min was performed to quench the IAA excess. Trypsin was added to the treated glycoprotein sample at a concentration ratio of 1:25 and incubated at 37 °C overnight. After incubation, the digestion was quenched at 90 °C for 15 min. In a second enzymatic digestion, Glu-C was added to the tryptic-digested sample at a concentration ratio of 1:25 and incubated at 37 °C overnight. The sample was finally dried down in a SpeedVac concentrator.The purified serum haptoglobin was resuspended with 20 μL of 50 mM NHm/z and a MS2 scan range of 300–3000 m/z were applied to a pooled sample for the identification of the precursor ions, their fragments and retention times. All the identified precursor ions were included in the PRM-MS method with a retention time window of ±3 min, and a mass range of ±2 Da relative to the target mass. In order to compare the data from two cohorts, t test and Wilcoxon test were performed and p-values < 0.05 were considered significant, the p-values observed in the evaluated groups were corrected using Bonferroni correction . We obtained area under receiver operating characteristic curve (AUC) values, and heat maps using the SPSS® version 27 (IBM) software. PCA plots were obtained using the software MarkerView® version 1.3 (AB Sciex).The dried tryptic and Glu-C digests were resuspended in a solution of 2% acetonitrile (MeCN), 0.1% formic acid (FA). Five microliters of the reconstituted sample were injected onto a C18 trap column  for 10 min, and the samples were then transferred to an Aclaim PepMap C18 capillary column  using an Ultimate 3000 nanoUHPLC system . The flow rate was set to 300 nL/min with a temperature of 60 °C. Mobile phase A was an aqueous mixture with 2% of MeCN, and 0.1% of FA, while mobile phase B was a mixture of MeCN with 0.1% of FA. The analytical gradient was 100 min long, and started at 2% mobile phase B for the initial 10 min, then increased to 38% at 11 min. During the next 70 min the organic phase B gradually developed to 60%. Subsequently, it ramped up to 90% in a period of 3 min and was maintained for 4 min. Finally, the percentage of organic phase B dropped to 2% in 1 min and was kept at that condition to pre-equilibrate the system. The nanoUHPLC system was interfaced to a Q Exactive , and operated in positive ion mode for PRM-MS analysis. The fragmentation pattern of the precursor ions was evaluated with different collision energy (CE) levels and established as 25 eV. Then, a MS full-scan range of 300–2000 N-glycopeptides can be used to differentiate NASH cirrhosis and NASH-related HCC. This assay incorporated a target parallel reaction monitoring (PRM) approach, with a long C18 column that was capable of resolving important isomeric glycopeptide structures at a high temperature. We were able to accurately quantify 72 isomeric structures corresponding to 42 glycopeptide compositions, which were structures that were distributed among the haptoglobin glycosylation sites as follows: thirteen in the site Asn184, forty-four in the site Asn207, and sixteen in the site Asn241. The glycosylation site NLFLN207HSE (Asn207) showed the largest number of glycan structures, most of them with isomeric forms. The microheterogeneity of this site was strongly related with the data ability to differentiate between the studied diseases, as can be observed in the PCA and heat map analysis. Additionally, quantitative analysis revealed four N-glycopeptides with significant changes in abundance between the cirrhosis and HCC samples (p < 0.05), all of them located in the site Asn207. Two of the significant N-glycopeptides were sialylated fucosylated structures that increased in abundance from cirrhosis to HCC. Two of the significant N-glycopeptides were tri-antennary sialylated structures that decrease in abundance from cirrhosis to HCC. The AUC values of the ROC curves were used to compare the accuracy of single and groups of N-glycopeptides in the differentiation of cirrhosis and HCC diseases. The results showed different group combinations with higher AUC values than AFP alone. Additionally, the N-glycopeptide Asn207 + 5-6-0-1 better differentiated between cirrhosis and early HCC than AFP.In this study, we revealed the site-specific microheterogeneity (isomeric composition) of serum haptoglobin, and demonstrated that the developed isomeric N-glycopeptides contributed to the enhanced differentiation between both diseases. Moreover, by applying our LC-PRM-MS strategy, we were able to identify single and groups of haptoglobin N-glycopeptides that have potential to differentiate between patients with NASH cirrhosis and NASH-related HCC.This study confirmed that site-specific glycoproteomic analysis is an important tool to evaluate serum haptoglobin changes between cirrhosis and HCC samples. Additionally, we showed that the unraveling of the haptoglobin isomeric


The East Winner Bank Shipwreck takes its name from the southern sandbank on Hayling Island near Portsmouth, UK. Examination of the wreck indicates a 19th-century carvel-built vessel. The sandbank is an active environment, meaning the wreck is rarely exposed to its full extent. Discussed here is work completed on the site before and during the social-distancing restrictions imposed by COVID-19. Documentary sources and previous detailed surveys suggest a possible identification for the wreck. The site appears to be an example of an everyday 19th-century coastal trading vessel, rarely explored archaeologically in the UK, with potential to contribute to discussions of the maritime technologies and maritime cultural landscape of regular folk. The investigation represents an excellent example of combining historical and archaeological data sets to further the interpretation of both sources, revealing details about the ship and its lasting impact on this stretch of coastline. Emerging from the sand of the East Winner Bank like the ribs of a great leviathan are the remains of a shipwreck Fig. . The seaThe 19th century was a period dominated by Britain’s global empire. In the century’s opening decade, the defeat of the French and Spanish navies at Trafalgar ensured there was no naval force that could contend with the dominance of the Royal Navy. This was followed shortly after by the defeat of Napoleon and the collapse of the French Empire, the only state that could challenge Britain. Britain’s maritime networks could not have functioned without Britain itself serving as the empire’s industrial heartland, producing and exporting consumer goods. That industry and its commercial network was sustained by coastal trade around the British Isles, moving raw materials, coal, and other supplies.A large sandbank is situated off the southwest corner of Hayling Island Fig. , frequenThe wreck itself is oriented along a north–south axis at the eastern edge of the sandbank. The maximum working time on the site is 30–45 min., depending on the depth of the tide, which turns fast and submerges the site relatively quickly. Even during very low tides (less than 0.8 m), the northern half of the site remains partially submerged. Interpretation of the site is based on the initial archaeological work conducted in 2014 and additional periods of fieldwork in 2017, 2019, and 2020.The site is composed of the remains of a carvel-built ship. The bow section is missing, with no stempost visible on the site, and the forward quarter of the ship has degraded to the second futtocks. The Maritime Archaeology Trust report of 2014 records that the remaining timbers on the site are in good condition overall and describes the wood as “fresh” . The hull elements consist of framing timbers, such as floor timbers, futtocks, and possibly top timbers , which has produced a comprehensive account of the archaeological and historical context of that site as well as an extensive summary of the archaeological involvement of multiple groups and methodologies. By collating the “Record” in this way, not only is this project able to offer a detailed interpretation of the shipwreck, but also to provide a single reference point for any ongoing work.Ultimately, the evidence contained within the wright’s  work on Water Nymph or Ossowski  and Ocean (lost in 1865) are strong candidates. Very few details about the third candidate, Johanna Elizabeth (lost in 1866), are recorded. However, that ship’s voyage at the time of wrecking was from Rio Grande  to Falmouth  carrying “bone dust.” Johanna Elizabeth is therefore likely to have been a merchantman engaged in international deep-ocean trade, a trade not suitable for a ship of less than 26 m in length and under 200 tons. Ocean and Fairy King, however, were documented as schooners carrying china clay, a raw material used in a wide range of industries, such as the production of porcelain. The china-clay trade was one of many that relied upon coasting vessels to move materials around the coast of Britain. Specific details of Ocean’s voyage are not listed in the Shipwreck Index of the British Isles; however, the cargo and vessel type suggest this ship is likely to have been one of the merchant schooners integral to Britain’s coastal trade.The shipwreck’s identity, therefore, has three remaining potential candidates, i.e., the “shortlist.” As schooners engaged in coastal trade, both Fairy King was not recorded as running aground in 1903. Reports were made of wreckage washing up along the Hampshire and Sussex coastlines. The first reports coming from Itchenor near Chichester Harbour  and then lost.our Fig.  reportedOcean’s wrecking, details emerge that seem to support its identification as the East Winner Bank Shipwreck. Of particular interest is the circumstance of the wrecking event. The ship was reported to be stranded on the East Winner Shoal within sight of the beach on Hayling Island. This correlates with the site of the ship today, which is located on the extreme eastern edge of the bank within site of the beach. Further details of the ship’s life that have been extracted from the Lloyd's Register   that the ship was built with iron knees. Furthermore, between 1836 and 1857 (excluding the 1852 and 1855 entries), the ship is recorded as being fastened with iron bolts. These details are not repeated in any of the other entries , 1823 (Cork), and 1832 (Cork). For most of Ocean’s life, the ship follows these regular patterns: registration at a survey port in Devon, then pursuing coastal trade around Britain and Ireland. There are, however, some journeys indicating one of the specific traits of merchant schooners  from Dartmouth. Trade with the Iberian Peninsula was particularly important to a port like Southampton, which had held the monopoly on the import of wine from Spain and Portugal for a long time  shows how these shares can work. In this case, Thomas Jones Stevens, a Plymouth shipbroker, acquires all 64 shares in the ship and then mortgages them to a different Thomas Stevens, a Plymouth-based merchant. The entire mortgage is paid off by Thomas Jones Stevens on 16 January 1857. All 64 shares are then sold to Thomas Stevens, the Plymouth merchant, on 21 January 1857. Finally, in 1861, Thomas Stevens sells his entire stake, 64/64, in an equal split to Thomas Jones Stevens and Sanders Stevens, both shipbrokers in Plymouth, 32/64 shares each. Thomas and Sanders Stevens are the owners of the ship when it is lost on the East Winner Bank on 18 January 1865. What this somewhat complex back and forth does show is the nuance hidden within the surname Stevens in the Lloyd’s Register ; Thomas Clark, aged 28, from Torpoint ; William Hooper, aged 55, from Mevagissey ; and William Geithard, aged 13, from either Stonehaven (Aberdeenshire) or Stonehouse (part of modern-day Plymouth). It is worth noting the master for this voyage is not the same man as the one recorded in the Lloyd’s Register for either 1862–1863 or 1863–1864, which was the vessel’s last registration (Lloyd’s Register Foundation Ocean again provides a glimpse of one of the important traits of Britain’s coastal trade and the advantage of a schooner-rigged ship; the crew did not need to be large for a voyage of this kind.Shipwreck Index of the British Isles reports that the ship was stranded on the East Winner Bank in a west-southwest force 9 wind (R. Larn and B. Larn Western Morning News on 17 January 1865 recounts the efforts to rescue the crew:In the Solent, the ship ran into trouble. The Western Morning NewsThe boat was launched as quickly as possible, Major Festing taking the helm, and after waiting some little time to allow the ebb tide to run out its greatest strength, the boat’s head was laid to the schooner, and the men bent lustily to their oars. It was a matter of life or death to all those in the boat, as it was to the expectant men in the unfortunate schooner’s rigging. (Standard on Wednesday, 18 January 1865, five days after the wrecking event: “January 16, the Ocean, Schooner, ashore in the Woolsteners, was bound for Newcastle, reported in yesterday’s list. She is embedded in the sand up to her bend; her masts are still standing and endeavours will be made as soon as the weather moderates to save materials” (StandardThe half-yearly accounts record two crewmen lost during the wrecking event: William Hooper and William Geithard, the ship’s boy. The description of the wrecking event and the resulting position of the ship correlate with the location and orientation of the site today (National Maritime Museum The description of the site being embedded “up to her bend” may be another way of referencing the turn of the bilge, which is how the site appears today, though more collapsed in its current state. The shipwreck left a lasting impact on this stretch of coastline. Following Major Festing’s rescue of the three surviving crew, he was awarded a medal for his heroics. A lifeboat station was also established on Hayling Island, which is today the site of the Hayling Island Royal National Lifeboat Institution.Ocean is an example of a type of ship that is largely absent from archaeological investigations of the 19th century in the UK. No previous investigation has looked at a ship of this type in the detail facilitated by the approach outlined in this article. As a working merchant schooner, Ocean was part of a system of coastal trade and connections that formed the basis of a wide-ranging global trade network. The degree of preservation is another reason this site has potential to make a significant contribution to the understanding of the 19th-century maritime world. The information obtained from the site so far directly informs understanding of the technologies used in 19th-century ship construction. That understanding is further improved by the information contained within the documentary evidence from the Lloyd’s Register of Shipping and the Plymouth Shipping Register.The schooner Ocean did not cease to impact the world when they were lost. They retain an ability to do so (Pétursdôttir Concurrently incorporating the analysis of archival documents with analysis of the archaeological material is an area in which the site can contribute to archaeological methods. This shipwreck, because of its status as an everyday working vessel, is a superb candidate to test and further develop such a methodology. By analyzing these documents as parts of the ship’s entire material assemblage, archaeologists can begin to understand the impacts that the individual components have on each other and the wider world. Ships like The documentary evidence also suggests a single candidate for the identification of the shipwreck and sheds light on its story. The site’s identification is relatively secure, as there are virtually no other candidates to which this shipwreck can be attributed.Ocean’s wrecking prompted the establishment of a lifeboat station, a facility that is still in operation today. This is also a story of ordinary people, not gigantic stock companies or elite individuals. Those individuals involved in Ocean’s everyday life were merchants, sailors, fishermen, an artillery major, shipbuilders, and a boy from Aberdeenshire. Through studying Ocean, the way the ownership of ships in this period was structured, the networks of trade and exchange around the coast of Britain, and even wider connections to Europe can be seen.The ship’s story can be extracted from the wreck, the register books, and other associated documentation, and reveals the site’s significance to the local area of Hayling Island. Undoubtedly, the site would benefit from further investigation. The work done to date created an overview site plan and established the material components of fastenings on the site. The next stage of work will be to examine the timbers and, if possible, conduct a more detailed and thorough exploration of the site. Future fieldwork will answer questions about the construction and specifics of the repairs made to the ship. As studies of ships of this kind in Britain are uncommon, there may not be a better opportunity to contribute to the understanding of the 19th century from such an overtly maritime perspective. This site allows archaeologists to examine the lives and stories of people who are all too often missing from or lost in the “Record.”


We show that rosin soap can inactivate human enveloped viruses: influenza A virus (IAV), respiratory syncytial virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For IAV, rosin soap could provide a 100,000-fold reduction in infectivity. However, rosin soap failed to affect the nonenveloped encephalomyocarditis virus (EMCV). The inhibitory effect of rosin soap against IAV infectivity was dependent on its concentration but not on the incubation time or temperature. In all, we demonstrate a novel chemical inactivation method against enveloped viruses, which could be of use for preventing virus infections in certain settings.Chemical methods of virus inactivation are used routinely to prevent viral transmission in both a personal hygiene capacity but also in at-risk environments like hospitals. Several virucidal products exist, including hand soaps, gels, and surface disinfectants. Resin acids, which can be derived from tall oil, produced from trees, have been shown to exhibit antibacterial activity. However, whether these products or their derivatives have virucidal activity is unknown. Here, we assessed the capacity of rosin soap to inactivate a panel of pathogenic mammalian viruses IMPORTANCE Viruses remain a significant cause of human disease and death, most notably illustrated through the current coronavirus disease 2019 (COVID-19) pandemic. Control of virus infection continues to pose a significant global health challenge to the human population. Viruses can spread through multiple routes, including via environmental and surface contamination, where viruses can remain infectious for days. Methods for inactivating viruses on such surfaces may help mitigate infection. Here, we present evidence identifying a novel virucidal product, rosin soap, which is produced from tall oil from coniferous trees. Rosin soap was able to rapidly and potently inactivate influenza virus and other enveloped viruses. Even before the current pandemic engendered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 19 (COVID-19), respiratory-borne viruses were a leading cause of global morbidity and mortality . StrategThe infectious particle of many respiratory viruses is encased in a phospholipid bilayer or “envelope,” which is essential for infectivity . For inf12–During a pandemic, there is likely to be an increased demand for products that eliminate viral infectivity from surfaces. Coniferous trees and some other plants produce liquid resin, which seals wounds in tree bark and protects the plant against pathogens and herbivores. Coniferous rosin contains resin acids, such as abietic acid and dehydroabietic acid, which are lipid-soluble diterpenoid carboxylic acids . Resin aNovel solutions for disrupting the transmission of human pathogens are required. Given that it exhibits antibacterial activity, we hypothesized that rosin soap may inhibit the transmission of pathogenic human viruses by killing the viruses on surfaces. We thus took rosin soap and assessed whether it could reduce the infectivity of IAV (strain WSN). We chose IAV because it is a model enveloped human RNA virus and is a significant human pathogen. Furthermore, IAV achieves high titers during propagation in cell culture and is highly cytopathic (rapid cell death and rounding) in traditionally used cell lines such as Madin-Darby canine kidney (MDCK) cells, which together allow the facile and sensitive determination of high levels of inhibition to viral infectivity. To determine whether rosin soap powder could reduce the infectivity of IAV, we incubated influenza virus stocks with rosin acid (2.5% [wt/vol]) at 37°C for 30 min and measured the residual infectivity using a limiting dilution assay, assessing the cytopathic effect 72 h later, in comparison to virus- and Dulbecco’s modified Eagle medium (DMEM)-only controls. In these initial experiments, incubation of IAV with rosin soap powder gave at least a 10,000-fold reduction in infectivity .Precluding a precise determination of the reduction in infectivity is the relatively high limit of detection in this assay, due to the cytotoxic effect of residual rosin soap on cell viability, which is necessary for the detection of IAV infectivity. Throughout our studies, we were hindered by the relatively high cytotoxicity of rosin acids at the maximum concentration on the cells used to measure residual viral infection. This relatively high limit of detection prohibited us from determining whether there existed any viral infectivity remaining. To decrease the cytopathic effect and thus reduce the background, we filter-purified our virus/rosin acid preparations prior to infectivity measurements. The experimental conditions were room temperature (RT) for 5 min. These experiments demonstrated a removal of the background cytotoxicity and the lower limit of detection: Enhanced virucidal activity against IAV  was observed . These data suggest that rosin soap very likely can inactivate all infectious virus particles in each sample at 2.5% (wt/vol), although we cannot formally prove this.Given its effect on IAV infectivity, we hypothesized that rosin soap may also inhibit other viruses. To this end, we investigated the virucidal activity of rosin soap against another IAV strain (the H3N2 strain Udorn), RSV, and SARS-CoV-2, as well as the nonenveloped EMCV. RSV and SARS-CoV-2 are representatives from two groups of viruses, the pneumoviruses and the coronaviruses, and are themselves significant human pathogens. EMCV is a model nonenveloped virus and a pathogen of pigs and other mammals, such as nonhuman primates. We carried out the same protocol as used above for IAV WSN and measured the residual viral infectivity using virus-specific means. The conditions for these experiments were room temperature for 5 min. In this series of experiments, all enveloped viruses were inhibited by rosin soap, although to different degrees, demonstrating that the activity of rosin soap is not limited to WSN or IAV . In all To understand more about the physiochemical dependence of the potent activity exhibited by rosin soap against enveloped viruses like IAV, RSV, and SARS-CoV-2, we next determined the effect of the rosin soap concentration, temperature, and incubation time on its virucidal activity. All previous experiments were carried out with a concentration of 2.5% (wt/vol), a time of 5 min, and at room temperature, so here, we decided to alter the concentrations , together with the incubation times  and incubation temperatures . Across all experiments, the virucidal activity of rosin soap was only dependent on the concentration, with 2.5% showing seemingly complete activity against IAV and a reduction in inhibition observed for each reduction in concentration . In contInfectious pathogenic human viruses, including SARS-CoV-2, can persist in the environment for extended periods of time, facilitating transmission via direct contact and/or through environmental contamination . StrategCritically, we investigated the limits of the virucidal activity of rosin soap by altering the temperature, concentration, and time of incubation. Using IAV as a model enveloped virus, this virucidal effect was dependent on the concentration of the product rather than the incubation temperature or time. Interestingly, while the virucidal activity of rosin soap was not influenced by the length of exposure (5 to 30 min) or incubation temperature , the only factor that did influence the virucidal activity was the rosin soap concentration, with 2.5% (wt/vol) being the most effective. Higher concentrations of rosin soap led to the rapid and potent loss of infectivity of IAV and other enveloped viruses. The fact that neither temperature nor time had a major impact on the efficacy suggests that this product has highly potent virucidal activity.Mechanistically, our results showing the lack of efficacy against nonenveloped viruses suggests that the target for the antiviral activity of rosin soap is the viral envelope, which is composed of a phospholipid bilayer. The virus envelope is critically required for infectivity, facilitating protection of genomic material and facile entry  into target host cells via virion-to-cell membrane fusion, either at the plasma membrane or endosomal compartment membranes . Loss ofin vitro cell culture conditions, we were not able to completely negate this background toxicity in our virus infectivity assays (which rely upon cellular integrity), even following the purification of our virus/soap mixes by filtration. However, our data suggest that it is highly likely that rosin soap inactivates the vast majority of infectious particles in a given preparation. Using IAV, which grows to very high titers, we were able to demonstrate nearly complete inactivation. It is worth noting that the level of virus titer used in these experiments is higher than that likely present in most “real world” scenarios/environments  in our ronments . Despiteronments .The virucidal activity of rosin soap when viruses are dried onto surfaces is an area that needs further research, as viruses such as SARS-CoV-2 persist on surfaces and are a source of infection transmission . This woIn conclusion, we demonstrated the virucidal activity of rosin soap against multiple pathogenic human enveloped viruses.2) and passaged routinely.Mammalian cell lines (MDCK [Madin-Darby canine kidney] and Vero [African green monkey] cells) were cultured in DMEM (high glucose) supplemented with fetal bovine serum (5% [vol/vol]) and penicillin/streptomycin (1% [vol/vol]). The cell cultures were maintained in flasks , respiratory syncytial virus (RSV-A2), SARS-CoV-2, and EMCV, were prepared using standard virology techniques on Vero (SARS-CoV-2 and EMCV), MDCK (influenza A virus), and Hep-2 (RSV) cells. For culture of IAV Udorn, serum-free medium was used, supplemented with TPCK -treated trypsin (Sigma-Aldrich) at a concentration of 1 μg/mL. The infectious stocks were produced and titrated in their respective cell lines before use in the virucidal activity experiments. All virus work was carried out in the biological safety level 2 (BSL2) or BSL3 (SARS-CoV-2) facilities at QUB.Pinus sylvestris L. and Picea abies L. The most abundant resin acid types included abietic acid, dehydroabietic acid, pimaric acid, and palustris acid. The pH of the raw undiluted product was pH 8 to 9, while diluted in DMEM at an antiviral concentration, it was pH 7 to 7.5, as estimated using pH indicator strips.The rosin soap was produced from crude tall oil by Forchem Ltd. . It was a water solution obtained from dried rosin salt, consisting of less than 10% sodium salts of tall oil fatty acids and over 90% sodium salts of resin acids. The resin acids and fatty acids of the product originated from the coniferous trees 2O). The negative control contained no virus and was incubated at 37°C. To each well of the first column, 100 μL of treatment and 100 μL of virus was added. After exposure to the experimental conditions, which were time  and temperature , 10-fold serial dilutions were carried out. Following dilution of the virus, permissive cells were added (100 μL) and incubated for 2 to 3 days. Viral infectivity was measured as the reciprocal of the final dilution showing a cytopathic effect, following manual investigation with a light microscope.The virus inactivation assays were carried out in 96-well plates. Initially, complete DMEM (100 μL) was added to each well, except the first column, which was used to incubate the virus and product. Three concentrations of rosin soap powder were tested under each condition in duplicate . Rosin soap  was dissolved in double-distilled water  were used. One hundred microliters of virus (WSN) was added to 100 μL of rosin soap (2.5% [wt/vol]) for 5 min at room temperature. The 200 μL (WSN/rosin soap powder) was washed through the filter units four times with 12 mL fresh DMEM . Tenfold serial dilutions were carried out. Following dilution of the virus, permissive cells were added (100 μL) and incubated for between 2 and 3 days. Viral infectivity was measured as the reciprocal of the final dilution showing a cytopathic effect.