Molecules that stabilizeprotein–protein interactions (PPIs)are invaluable as tool compounds for biophysics and  biology,and as starting points for molecular glue drug discovery. However,identifying initial starting points for PPI stabilizing matter ishighly challenging, and chemical optimization is labor-intensive.Inspired by chemical crosslinking and reversible covalent fragment-baseddrug discovery, we developed an approach that we term “molecularlocks” to rapidly access molecular glue-like tool compounds.These dual-covalent small molecules reversibly react with a nucleophilicamino acid on each of the partner proteins to dynamically crosslinkthe protein complex. The PPI between the hub protein 14-3-3 and estrogen-relatedreceptor γ (ERRγ) was used as a pharmacologically relevantcase study. Based on a focused library of dual-reactive small molecules,a molecular glue tool compound was rapidly developed. Biochemicalassays and X-ray crystallographic studies validated the ternary covalentcomplex formation and overall PPI stabilization via dynamic covalentcrosslinking. The molecular lock approach is highly selective forthe specific 14-3-3/ERRγ complex, over other 14-3-3 complexes.This selectivity is driven by the interplay of molecular reactivityand molecular recognition of the composite PPI binding interface.The long lifetime of the dual-covalent locks enabled the selectivestabilization of the 14-3-3/ERRγ complex even in the presenceof several other competing 14-3-3 clients with higher intrinsic bindingaffinities. The molecular lock approach enables systematic, selective,and potent stabilization of protein complexes to support molecularglue drug discovery. For instance, chemicalcrosslinkers,4 genetically encoded reactivehandles,6 and photo7/chemical9-affinity labels have provided a deep mechanistic understanding ofthe proteins under study. Moreover, covalent chemical probes, tetheredfragment libraries,12 ligand-directed cargo release,13 and covalent targeted protein degraders15 have enabled the study of disease states and have rapidly empowereddrug discovery.Covalent tool compounds are instrumentalin investigating proteinfunction, proteomics, drug discovery, and protein visualization.17 IMiDs,20 dCeMMs,21 and EN45022 haveprovided a greater understanding of protein–protein interaction(PPI) regulatory mechanisms and disease dysregulation.23 Moreover, native PPI stabilizers like NRX-10309424 and Trametiglue25 stand as compelling examples of the potential of native proteincomplex stabilization. Notwithstanding, the current paucity of molecularglues and of conceptual approaches to identify molecular startingpoints for their development highlights the significant challengesin designing “glue-like” chemical probes. This challengeat least partially originates from a lack of native ligands and thedynamic nature of multicomponent protein complexes. Consequently,covalent tool compounds could provide entries to study and “drug”a greater scope of PPIs.Molecular glues have also emerged as a powerfulchemical biologyconcept and drug discovery innovation. These small molecules stabilizeprotein complexes leading to enhanced protein complex avidity. Molecularglues like Rapamycin,22 andRM-18,27 which stabilize the UBE2D/NFκB andKRASG12C/CYPA protein complexes, respectively, demonstratethe promising role of reactive handles in molecular glue tool compounddesign. Covalent chemical probes react specifically with a nucleophilicamino acid on a protein surface using an electrophilic moiety, suchas an acrylamide or β-haloketone group. This is beneficial asthe off-rate of small-molecule binding is reduced, enhancing targetengagement. The targetable scope of nucleophilic amino acids has moreoverexpanded beyond cysteine, to encompass serine,28 lysine,31 and arginine,32 providinggreater control of anchoring sites on the protein and expanded possibilitiesfor more diverse tool compounds that stabilize protein complexes.Covalent drug discovery has already shown significant success in targetingchallenging proteins,36 which makes the strategy promisingto molecular glue tool compound development.Covalent molecular glues EN45038 disulfide,41 α-ketoamide,43 and aldehyde31 electrophiles has further diversified the portfolio and propertiesof chemical probes and tool compounds in chemical biology. The exchangeablenature of reversible covalent tethering is similarly attractive formolecular glue design. We and our colleagues previously developedreversible covalent fragment tethering approaches to identify initialchemical matter to stabilize various protein complexes involving thehub protein 14-3-3.44 Disulfide and iminetethering was instrumental in identifying fragments for stabilizationof the 14-3-3/ERRγ and 14-3-3/p65 protein/phosphopeptide complexes,respectively , differential scanning fluorimetry(DSF), and peptide washout experiments provided insight into the mechanismof protein–complex stabilization. Specifically, molecular lockbinding is driven by the interplay of molecular reactivity and recognitionby the composite PPI binding interface. The molecular lock approachis highly selective as illustrated by the stabilization of the 14-3-3/ERRγcomplex in the presence of several other competing high-affinity 14-3-3client peptides and supported by a mathematical model. Further, celllysate experiments showed that the approach is applicable to the reactiveenvironment of the cell. The dual-reactive molecular locks provideunique and rapid opportunities for selective PPI stabilization.Utilizing the interaction between the hub protein14-3-31 in complex with 14-3-3/ERRγ45 and fragment 2 in complex with14-3-3/NF-κB51  . Locks 5–6 and 9–10 were designed basedon previous chemistry campaigns, which identified that halogen-substitutedphenyl rings probe a subpocket of 14-3-3 shaped by residues Ser45,Phe119, and Met123, resulting in enhanced stabilization.53 Additionally, the amide bond (τ = 30 and 150°) was replacedwith a sulfonyl amide, 7, (τ = 90 ± 30°)to investigate the significance of torsion angles between the carbonyl/sulfonyland beta-carbon of the linker.Seven dual-reversiblecovalent molecules were developed to investigate molecular lockingof the 14-3-3/ERRγ protein complex 1d. The d/NF-κB51 1. Previoine bond 1a,b. Anag (∼4 Å) 1e, which4–6 and 8–10 were accessed via a three-step synthesis.Starting from an amide coupling of the benzoic acid with cystamineHCl, subsequent N-methylation and disulfide exchange with 2,2′-bis(dimethylamino)diethyldisulfide dihydro chloride afforded the compounds in acceptable yield(Scheme S1). Compound 7 wassynthesized via an amide coupling from the diacetate sulfonyl chlorideprecursor followed by deprotection, N-methylation, and disulfide exchange(Scheme S2).Compounds Figure S1). Complex stabilizationafter a 24 h incubation was quantified by dividing the KD of the binary 14-3-3/ERRγ complex (DMSO control)by the apparent KD (KDapp) of the 14-3-3γ/ERRγ complexupon stabilization with 100 μM of each compound, providing thestabilization factor (SF100) (Tables S1 and S2). Notably, we define KDapp as the binding affinity of the ERRγ peptide to 14-3-3γin the presence of a stabilizer.The stabilization of the ERRγ/14-3-3γcomplex by themolecular locks was assessed using a fluorescence anisotropy (FA)assay where 14-3-3γ was titrated into a fluorescently labeledERRγ phosphopeptide in the absence or presence of a 100 μMmolecular lock 2a,b; thi1 and 4–10 all induced a leftward shift of the binding curve comparedto thebinary 14-3-3γ/ERRγ complex (KD = 6.5 ± 0.9 μM), indicating stabilization of the PPI(Table S1). Compound 1, containingonly the disulfide moiety, elicited a five-fold stabilization of the14-3-3γ/ERRγ complex (KDapp = 1.4 ± 0.2 μM), and compound 3, containing only the aldehyde moiety, elicited no protein complexstabilization (SF100 = 1). The dual-reactive molecularlocks showed a significant increase in the stabilization factor, withthe SF100 ranging from 7.3 to 37 (14-3-3γ/ERRγ KDapp = 0.92–0.18 μM).Interestingly, the 14-3-3/ERRγ composite pocket favored an m-substitution of the reactive handles compared with a p-substitution (8 vs 4). Substitutionof the amide for a sulfonyl amide showed no significant change inthe stabilization. (4 vs 7).Halogen-substituted benzamide rings improved stabilization , except for 9. The optimal combination proved to be a 3,5-substitutionof the bromine and aldehyde functionalities (10).Compounds Figures S2 and S3). All fragmentsreached saturation by ∼12 h. Disulfide 1 showedsignificantly slower binding kinetics than molecular locks 4–10. Carboxamides 4–6 and 8–10 reached saturationwithin 5 h. Molecular lock 5 elicited the fastest reactionkinetics, with saturation reached at 2 h. Interestingly, 5 also elicited the second lowest KDapp value.Time-dependent fluorescence anisotropy (TD-FA) measurements providedinsights in the kinetics of ternary complex (14-3-3/ERRγ/lock)formation  complex,and the ternary complex of 14-3-3γ, the ERRγ phosphopeptide,and compounds 1 and 3–10 . The DSF data corroboratedthe enhanced stabilization observed in the FA data, with molecularlocks 5 and 10 eliciting 14-3-3γ meltingtemperature increases of 4.7 ± 0.2 and 6.0 ± 0.3 °C,respectively , and compound 3 induced no significantchange. Interestingly, TD-FA and DSF assays showed that while allcarboxamide-based molecular locks elicited similar binding kinetics,a clear difference in the final avidity of the stabilized complexwas observed. This result suggests that stabilization of the complexis driven by tripartite molecular complementarity between the molecularlock, ERRγ, and 14-3-3, not simply reactivity.Differential scanning fluorimetry (DSF) measurementsrevealed the effect on the thermal stability of the 14-3-3/ERRγPPI complex by the molecular locks. We screened the ectively 2c. In co9, in cocrystallization with the ERRγ phosphopeptide and 14-3-3σ(C38N) . The 14-3-3σisoform was used for the cocrystallographic studies because of itsfavorable crystallization properties. 14-3-3σ features a cysteine(Cys38) in the binding groove in proximity to 14-3-3 Lys122. The cysteinewas mutated into an asparagine (C38N), as present in the other 14-3-3isoforms, to avoid potential reactivity in the crystal structures.The inability of compound 9 to cocrystallize was in linewith the FA studies where 9 was the least active. Forall other molecular locks, a continuous density was observed for theentire compound, including both the covalent bonds with Lys122 of14-3-3 and Cys180 of ERRγ . In addition to the dynamic covalent bonds, the compounds alsomake noncovalent interactions with both 14-3-3 and with ERRγ,responsible for the tripartite molecular complementarity between themolecular lock, ERRγ, and 14-3-3.High-resolution X-ray structures (<1.4 Å)were obtained for all molecular locks, except compound 5 and the 14-3-3γ/ERRγ/10 protein complexes. To ensure detection of the acid-labileimine-bonded complexes, the covalent imine bond was reduced in situ to the corresponding secondary amine using sodiumcyanoborohydride. Samples were incrementally taken over 4 h and analyzedwith high-resolution MS to determine crosslinking of the 14-3-3γ/ERRγcomplex . Intact MS analysisconfirmed time-dependent crosslinking of the ternary complex (14-3-3γ/ERRγ/lock).Treatment with 5 resulted in near quantitative consumptionof apo-14-3-3γ into the 14-3-3γ/ERRγ/5 complex with a molecular weight of 33,019.9 Da after 4 h.Notably, ternary complex formation was not observed in the absenceof sodium cyanoborohydride .A similar result was also observed for 10  . Notably, although compound 10 eliciting improvedstabilizer activity in FA assays, complete consumption of apo-14-3-3γ was not observed.To further confirm thecovalent crosslinking in solution, we performed intact mass spectrometry(MS) on the 14-3-3γ/ERRγ/We hypothesizethe enhanced avidity of the ternary complex to be a function of themultivalent binding of the dual-reactive groups. Specifically, thedissociation of a molecular lock must pass through two equilibriumreactions leading to high effective molarity of the compound at theprotein–protein interface and a high energy barrier for thedissociation of the ternary complex.1 and molecular locks 5 and 10 were screenedin FA and DSF assays in the presence of either 50 μM or 1 mMTCEP. Previous research has shown that low concentrations of reducingagents can enhance disulfide exchange.10 In contrast, a high concentration of the reducing agent resultsin a complete reduction of disulfide bond formation. Analysis of theFA data showed stabilization by compounds 1, 5, and 10 at low TCEP concentrations (50 μM)  was completely abolishedat a high TCEP concentration of 1 mM . A similar effect was observed in DSF, where the increase in meltingtemperature upon addition of the molecular locks was abolished inthe presence of high concentrations of TCEP , indicating the need for the disulfide in the dual-covalentlock to stabilize the ERRγ/14-3-3γ complex. The importanceof the disulfide was further confirmed in FA assays using an ERRγC180S mutant .To test the effect of dualreactivity on PPI stabilization, the control compound Figures S14–S16). Analysis of the thermodynamicparameters showed that pH did not affect the KD of the binary 14-3-3/ERRγ complex (DMSO control) . Similarly, the avidityof the 14-3-3/ERRγ/1 complex at equilibrium wasnot affected by pH , connected to impairedimine bond formation at low pH. Notably, at pH 6.5, molecular locks 5 and 10 even showed similar stabilizing capacityto disulfide 1, indicating the strong enhancement ofthe PPI stabilization by the molecular locks upon imine bond formation.TD-FA experiments were also performed at differentpH values tostudy ternary complex (14-3-3γ/ERRγ/compound) formationand the importance of the imine bond in the molecular lock . Interestingly, whereasthermodynamically, the 14-3-3γ/ERRγ/1 complexformation was pH-independent, kinetic analysis clearly indicated thatthe speed of compound 1-induced PPI stabilization waspH-dependent. At pH 6.5, the kinetics of disulfide bond formationwas 50% slower. For instance, at pH 6.5, the maximum effect of 1 was reached after a 4.2 h incubation, compared to 1.7–0.8h for pH 7.0–8.0 .Similar trends were observed for molecular locks 5 and 10, where time-dependent complex stabilization was directlyaffected by pH, with low pH impeding ternary complex formation . Effective disulfide formationthus appeared to aid in the imine bond formation of the dual-covalentlocks. Combining the two reactive handles increased the effectivemolar concentration of the lock within the binding pocket, illustratingthe need for dual reactivity and leading to enhanced 14-3-3/ERRγavidity.Kinetic analysis showed that binary 14-3-3γ/ERRγ complexformation occurred rapidly, independent of pH, and remained constantover time . The beadswere then washed five times in a peptide and compound-free buffer.During each wash step, the beads were incubated in the buffer for20 min to allow dissociation of the ERRγ peptide from the immobilized14-3-3γ. The fluorescence was then measured for all samplesbefore capture, after capture, and after each washing step , and the amount of peptide recoverywas calculated. Notably, the final data were normalized against recoveryof the ERRγ peptide in the absence of biotinylated 14-3-3γto correct for nonspecific ERRγ binding . Analysis of the washout experiment showed asignificant reduction in the total recovered ERRγ peptide fordisulfide fragment 1 and dual-reactive molecular lock 5, with 58 and 44% peptide recovery, respectively (1 and 5 (63% for DMSO vs 42% with 1 and 36% with 5), indicating kinetic stabilization of 14-3-3γ/ERRγcomplex. Subsequent washing steps showed significantly more peptiderelease for 1 compared to 5. Notably, afterfive washes, incomplete recovery of the ERRγ peptide was observedfor 5. These results indicate that the presence of asecond reactive handle slows down the dissociation of the ternarycomplex as the molecular lock must pass through two equilibrium reactions.Next,a washout experiment was performed to understand ternary 14-3-3/ERRγ/molecularlock complex dissociation. Fluorescein-labeled ERRγ was incubatedovernight with biotin-functionalized 14-3-3γ in the presenceof either DMSO, disulfide ic beads S17a. Theic beads S17a. The54 We hypothesized thatthe interplay of chemical reactivity, the topology, and functionalityof the composite binding pocket shaped by 14-3-3 and the interactionpartner drives selective stabilization of the 14-3-3/ERRγ complex.53 To validate selectivity, molecular locks 5 and 10 were tested against a panel of nine14-3-3 client peptides in an FA assay, and the SF100 wasdetermined for each 14-3-3 PPI . This client panel contained a diverse representation of 14-3-3binding proteins, differing for example in size and hydrophobicityof the +1 amino acid. Importantly, clients PKR and RND3, each containinga +1 Cys, were included in the panel.Drugging the hub protein 14-3-3 raisesthe challenge of selectivity, given that 14-3-3 binds several hundredphosphoprotein partners 4a.54 We 5 and 10 provedhighlyselective for the 14-3-3/ERRγ complex in the client screen .Molecular locks t screen 4b. Molec1, locks 5 and 10 werealso screened against PKR and RND3 in the TD-FA assay. In contrastto binding to ERRγ, fragments 1, 5, and 10 showed no stabilization of the PKR complex. We reasoned that 10 adopted an unfavorableconformation for crosslinking, potentially a function of the stericclash between +3 Val of RND3 and 10 that destabilizedthe interactions. In ERRγ, a less sterically bulky +3 Ala ispresent , molecular lock 5 (10 eq.), ΕRRγ (2eq.), and eight additional client phosphopeptides (8 × 2 eq.)was incubated, and incremental samples were taken, quenched with sodiumcyanoborohydride, and measured  , with a maximum crosslinking efficiencyof 35% and no evidence of any other crossed linked 14-3-3 PPI. Remarkably,given the stoichiometry of peptide competitors relative to ERRγ(8:1), only an ∼2-fold reduction in crosslinking capacity wasobserved compared to the 14-3-3/ERRγ in isolation (∼70%)5e.55 The modelderived the fraction of protein complexes based on the concentrationsof all species used in the intact MS experiment , and peptide affinities to 14-3-3 were based on FAstudies .To gain a thermodynamic understanding of 14-3-3/ERRγ complexformation in the competitive environment, a multicomponent equilibriummodel was constructed. The model contained the mass balance and equilibriumequations of a system where one species A (14-3-3) can individuallybind to nine other species B–J (partner peptides) with connecteddissociation constants Kd1–Kd9 5f.55 TheFigure S24) unsurprisingly showed that peptide affinity dictated thepercentage of total 14-3-3 occupied. The highest-affinity peptide,PKR (KD = 180 nM), occupied 61% of thetotal 14-3-3 population, while the low-affinity peptide p65 (KD = 260 μM) occupied only 0.1% of thetotal 14-3-3 population. ERRγ occupied only 2.4% of 14-3-3,based on its KD of 6.5 μM. Comparisonof the fraction of ERRγ bound (2.4%) in the modeled data withthe intact MS result (35%) upon addition of molecular lock 5 indicates that 5 significantly increased the totalnumber of 14-3-3/ERRγ complexes in solution, by 15-fold.Analysis ofthe nonstabilized equilibrium model (5) formation, we also modeled the 14-3-3γ/ERRγmolecular lock-stabilized system . Here, the apparent KD of ERRγto 14-3-3γ was calculated, based on the intact MS-observed 35%complex formation and the KD values ofthe binary 14-3-3/peptide interactions. Notably, a key assumptionwas made that the other eight peptides were unaffected by the additionof 10 eq. of the stabilizer. The thermodynamic model calculated a KDapp of the 14-3-3γ/ERRγ/5 complex of 0.2 μM in the presence of 50 μM 5. Further analysis of the modeling data showed that the costfor enhanced 14-3-3 binding to ERRγ was paid by all other complexesroughly equally, with a 33–42% fractional reduction in occupancyof bound 14-3-3 complexes observed for all other client protein interactions.Notably, as a percentage, low-affinity binders contributed marginallymore to the increased ERRγ binding, with the low-affinity binderp65 having the largest fractional reduction (42%) in 14-3-3 bindingand the high-affinity binder PKR the lowest fractional reduction (33%).However, the strongest binders contributed the most of the 14-3-3required in terms of total quantity. It should be noted that, in contrastto the situation in a cellular setting, in our model experiment, thereis a limiting concentration of hub protein, challenging the redistributionof 14-3-3 with all its interaction partners. The natural large reservoirof 14-3-3 will ensure enough buffering capacity of the protein forfunctional locking of specific complexes and molecular glue action.Taken together, these results showed the functionality of selectivestabilization of a hub protein’s PPI in a competitive environment,with several high-affinity interaction partners in competition for14-3-3, with our molecular locks.To gain further insight into lock-induced ternary complex . Analysis of the binary 14-3-3γ/ERRγ PPI showed thatthe complex was inhibited by the lysate, potentially a result of acompetition with high-affinity 14-3-3 binding partners present inthe lysate. Next, we investigated molecular lock 5-inducedcomplex formation .Gratifyingly, the 14-3-3γ/ERRγ/5 complexwas observed up to 2 mg mL–1 cell lysate, albeitat a loss in affinity  of 240 μM). Moreover, early observed cell lysate-inducedabolishment of the 14-3-3γ/ERRγ complex formation wasalmost completely recovered upon addition of 5, to similarcomplex concentrations to that in the absence of the lysate. Thisis remarkable given the limitations of disulfide reactive groups ascellularly compatible electrophiles. The result of tool compound 5 shows the potential of this concept for the developmentof dual-covalent chemical probes with more cellularly compatible electrophiles,such as cyanoacrylamides, to investigate PPI stabilization in cells.The cell represents a complexbiochemical environment composed of a high reducing potential, a poolof potential nucleophiles, and competing 14-3-3 interaction partners.To investigate molecular lock-induced complex formation in a reactiveenvironment, we first investigated binary 14-3-3/ERRγ formationin the HEK293 cell lysate. Here, the HEK293 cell lysate was titratedto the preformed 14-3-3γ/ERRγ complex in an FA assay andincubated overnight (The design of molecular glues and identificationof chemical startingpoints for PPI stabilization remain a significant challenge for drugdiscovery. Novel approaches to develop tool compounds and chemicalprobes are needed, both to investigate the regulatory roles of PPIsand as chemical leads for aiding drug development. Here, we presentan integrative approach to develop “molecular glue-like”tool compounds from tethered fragments. We demonstrate that with arelatively small library of compounds, rapidly, a strong stabilizationof the 14-3-3/ERRγ PPI can be achieved using dual reversiblecovalent chemistry. Enhanced complex stability is a function of themultivalent binding of two reversible covalent handles and molecularrecognition of the PPI interface, which enhances the effective molarconcentration of the ligand within the composite binding pocket. Further,the molecular locks show remarkably selective stabilization of the14-3-3/ERRγ complex in a competitive environment of other multiple,stronger binding, 14-3-3 interaction partners. The data revealed thisselectivity to be a function of the interplay between reactivity andmolecular recognition of the molecular locks and PPI. A concomitantmathematical model helps to both understand the complex interactionequilibria in competitive mixtures and to determine resulting affinityconstants upon molecular locking of the PPI at hand. The molecularlock approach thus provides a rapid means of coarse-grain assessmentof a PPI target for molecular glue development.The conceptof molecular locks, as tool compounds for addressingPPIs, can be easily translated to other reactive handles, such ascyanoacrylamides or α-ketoamides. The recent expansion of targetablenucleophilic amino acids makes this concept modular and expandableto include a wide scope of potential PPI targets. The broad scopeof reactive handles allows the chemical properties of the dual-reactivemolecular locks to be tailored to the local environment of the bindingpocket or the PPI interface, potentially complemented by strategicallyintroduced point mutations for molecular targeting of the locks. Themolecular lock concept should not be limited to molecular glues andcould easily be implemented to other fields of drug discovery. Forinstance, this concept should be latterly transferable to covalentPROTACs with the introduction of a second reactive handle providingenhanced residence times of the E3 ligase protein complex throughenhanced effective molarity.


Such ionic ferroelectrics with the absorbed water molecules further present the controlled tunability in polarization from 0.68 to 1.39 μC/cm2, thermal conductivity by 13% and electrical resistivity by 86% due to the proton transfer in an ionic lattice under external stimuli. These findings enlighten the development of molecular ionic ferroelectrics towards multifunctionality.Molecular ferroelectric materials consist of organic and inorganic ions held together by hydrogen bonds, electrostatic forces, and van der Waals interactions. However, ionically tailored multifunctionality in molecular ferroelectrics has been a missing component despite of their peculiar stimuli-responsive structure and building blocks. Here we report molecular ionic ferroelectrics exhibiting the coexistence of room-temperature ionic conductivity (6.1 × 10 Molecular ferroelectrics contain stimuli-responsive structure and ionic building blocks, promising for ionically tailored multifunctionality. Here, the authors report molecular ionic ferroelectrics exhibiting the coexistence of room-temperature ionic conductivity and ferroelectricity. Molecular ferroelectrics are typically composed of ionic building blocks9, enabling a possibility of the flow of ions11. However, the displacement of charged ions tends to impede the electron flow in electronic-insulating ferroelectric lattice. An interest arises in materials science if molecular ferroelectrics could be simultaneously ionic conductors that enable the flow of charged ions in the lattice, deemed as molecular ionic ferroelectrics  for a prototypical exampled candidate14, a solid-state molecular ionic ferroelectric crystal, to show ionic conductivity and ferroelectricity, combining properties of conventional ionic conductors22 and ferroelectrics28  and perchlorate anion (ClO4−). Those cation and anion arrays are alternatively arranged and locate in (101) planes  at room temperature, the equilibrium states are stabilized by a spontaneous polarization that is corresponding to one of the degenerate energy minima in the double-well Landau free energy33  plane, presenting a set of X-ray diffraction (XRD) peaks that equally distributed in diffraction angles  stretching and (in-plane and out-of-plane) deformation modes34 of anionic ClO4− are clearly presented by Raman peaks  and imaginary (−Z″) parts with the slope of approximately 3 at the low-frequency region  loops  confirmed that ImClO4 undergoes an endothermic phase transition from ferroelectric to paraelectric at around 373 K by heating, and an exothermic transition at around 365 K by cooling , implying ferroelectric ordering induced polarization-controlled thermal conductivity38  and repeated cycles of controlled humidity (17 and 70%). Once ImClO4 is periodically switched between a low humidity and a high humidity states, its resistivity decreases by about 86%. Proton facilitates the change of electrical resistivity, indicating a modified electronic structure favored for a reduced resistivity because of proton-related coordination in lattice. Similarly, thermal conductivity shows a significant decrease when humidity increases from 17% RH to 70% RH  curves are measured and a sharp jump is found that is absent in the initial I–V curve of ImClO4  planes of ImClO4 crystal, revealing a coexistence of ferroelectricity and proton conductivity in molecular ionic-ferroelectrics.The tunability of thermal conductivity, polarization, and resistivity is ascribed to proton transfer in ionic ferroelectric ImClO4) as molecular ionic ferroelectrics with the coexistence of ferroelectricity and ionic conductivity, which simultaneously shows ionically controlled ferroelectric behavior. As an ionic ferroelectric with the absorbed water molecules in lattice, ImClO4 exhibits its flexible tunability in remanent polarization, thermal conductivity, and electrical resistivity by humidity and external biases. This study provides the understanding of ion/polarization/thermal/resistive properties of molecular ferroelectrics and opens an effective route to explore more bifunctional materials based on molecular ferroelectrics.In summary, molecular ionic ferroelectrics are shown as multifunctional materials that possess ferroelectricity and ionic conductivity in a single phase. We introduce imidazolium perchlorate  hysteresis loops were measured on a Precision LC Ferroelectric Tester with the addition of a high voltage interface and a Trek 609B high voltage amplifier . Humidity-dependent P-E loops were conducted by putting ImClO4 samples into a container where humidity was controlled by a humidifier. Electrical biases tuning and current-voltage curves were conducted on a Keithley 2450 SourceMeter. Once different resistive states were obtained, ImClO4 sample was switched into another circuit for ferroelectricity measurements. The relative permittivity was measured on a precision impedance analyzer (Agilent 4294 A). A high-voltage dielectric probe  was mounted onto a physics properties measurement system (PPMS) for temperature and magnetic field control.Polarization-electric field  was used to measure electrochemical impedance spectra of ImClO4 crystals were collected on an Agilent Cary 630 FTIR spectrometer. Humidity was controlled by a humidifier. Moisture was kept to blow onto sample stage until a certain humidity was reached.Transmittance spectra of ImClO4 crystals by X-ray diffraction. The surface morphology was measured on Carl Zeiss AURIGA (200 kV) Field Emission Scanning Electron Microscope (FESEM). The element analysis was determined by Oxford energy-dispersive X-ray spectrometer (EDS).A Rigaku Ultima IV  was used to characterize the crystal structures of ImClO4 crystals were collected on Renishaw inVia Raman Microscope. The excitation wavelength is 785 nm.Raman spectra of ImClOThe thermal conductivity was measured on ferroelectric powder and film samples by using a Hot Disk TPS 2200 instrument . Ferroelectric films were prepared by spin-coating saturated aqueous solutions on FTO substrates. Since the ferroelectric sample is spontaneously polarized at room temperature, the thermal conductivity measured during heating was starting from a polarized state. When cooling from high temperature, the sample was in a unpolarized state. The humidity-dependent thermal conductivity measurements were conducted at room temperature.4 from 176 to 381 K was detected using Differential Scanning Calorimetry .Heat flow of ImClO43 was used to measure laser-induced shock velocities, plasma emission spectra, and time-resolved infrared emission. Before measurement, humidified crystal has been placed in an atmosphere with a relative humidity of 54% for half an hour. Another pristine crystal was measured as a reference. Both samples were ablated, atomized, ionized, and excited by a 6 ns pulsed laser (1064 nm) with the power of 850 mJ or 180 J/cm2. The laser-induced shock velocities were collected using high-speed Schlieren imaging  and were used to estimate the detonation velocity of the material. A high-resolution echelle spectrometer equipped with an intensified charge coupled device detector  was used to collect the emission spectra. An IR-sensitive photoreceiver  was used to monitor the integrated emission from the combusting particles on the millisecond timescale. Data from 20 laser shots were collected from each sample.The LASEM-420 systemSupplementary informationPeer Review File


Kaposi sarcoma (KS) is a multicentric tumor caused by Kaposi sarcoma herpesvirus (KSHV) that leads to morbidity and mortality among people with HIV worldwide. KS commonly involves the skin but can occur in the gastrointestinal tract (GI) in severe cases.RNA sequencing was used to compare the cellular and KSHV gene expression signatures of skin and GI KS lesions in 44 paired samples from 19 participants with KS alone or with concurrent KSHV-associated diseases. Analyses of KSHV expression from KS lesions identified transcriptionally active areas of the viral genome.Fms-related tyrosine kinase 4 (FLT4), encoding an angiogenic receptor, and Stanniocalcin 1 (STC1), a secreted glycoprotein. FLT4 and STC1 were further investigated in functional studies using primary lymphatic endothelial cells (LECs). In these models, KSHV infection of LECs led to increased tubule formation that was impaired upon knock-down of STC1 or FLT4.The transcript of an essential viral lytic gene, ORF75, was detected in 91% of KS lesions. Analyses of host genes identified 370 differentially expressed genes (DEGs) unique to skin KS and 58 DEGs unique to GI KS lesions as compared to normal tissue. Interleukin (IL)-6 and IL-10 gene expression were higher in skin lesions as compared to normal skin but not in GI KS lesions. Twenty-six cellular genes were differentially expressed in both skin and GI KS tissues: these included This study of transcriptional profiling of KS tissue provides novel insights into the characteristics and pathogenesis of this unique virus-driven neoplasm.The online version contains supplementary material available at 10.1186/s12967-023-04517-5. Kaposi sarcoma (KS) is caused by Kaposi sarcoma herpesvirus  . In addiSimilar to other herpesviruses, KSHV infection generally exists either in a state of latency, in which a few of the viral genes are expressed and no infectious progeny are released, or lytic replication, in which most viral genes are expressed and infectious virions are produced and disseminate within and between hosts , 11. MosKS most commonly manifests as skin lesions but may also occur in visceral organs including the respiratory and gastrointestinal (GI) tracts in severe cases , 25. PatWe sought to understand how host gene expression and viral transcripts are altered in KS as compared to normal tissue using matched patient samples. In this study of participants with well-annotated clinical characteristics, we also investigated both skin and GI KS to define differences in the viral and host transcripts between these sites of these lesions. We examined differences in the immune microenvironment by location of the KS lesion or by specific clinical characteristics, such as the presence of concurrent KAD. Specific cellular genes of interest that were identified from patient samples were further investigated using KSHV-infected lymphatic endothelial cells (LECs) infected to determine their pathological significance.Individuals with KS under the care of the HIV and AIDS Malignancy Branch at the National Cancer Institute were included. Clinical and HIV characteristics were obtained at the time of biopsy collection. KSHV viral load (VL) in peripheral blood mononuclear cells (PBMCs) was assessed by quantitative real-time polymerase chain reaction as previously described .Participants had a 6 mm punch biopsy of cutaneous KS over the lower limb, with normal appearing skin obtained within the same limb. If participants had gastrointestinal symptoms, an endoscopy or colonoscopy was performed and the operator visually identified KS lesions that were biopsied. An adjacent area of normal mucosa was obtained at the same time. KS biopsies were divided, and a portion was sent to the laboratory of pathology for histological confirmation. Two samples (G4T and G5T) were biopsied as abnormal mucosa in patients with known skin KS but on analyses, these lesions did not have detection of KSHV LANA by immunohistochemistry staining. However, these lesions did have KSHV transcripts detected by RNA-sequencing and thus were included in these analyses. All participants were consented to protocols for tissue procurement (NCT00006518) and/or sequencing of KS and other KSHV-associated diseases (NCT03300830) to permit RNA sequencing of KS lesions for this study. Both protocols were approved by the NCI Institutional Review Board. All enrolled participants gave written informed consent in accordance with the Declaration of Helsinki.cutadapt  #ab8245 (AbCam). Blots were scanned on an Odyssey scanner (Li-Cor).HDLEC cells were transfected with siRNAs, then infected with KSHV BAC16 at MOI of 0.25. At 1 dpi, cells were split to keep them sub confluent. At 2dpi, cells were harvested, counted, and seeded in duplicate at 0.3 million cells/mL in 96-well plate on Cultrex Ultimatrix Reduced Growth Factor Basement Membrane Extract (Bio-Techne #BME001-05) for tube formation assays using the thick gel method. At 16 ht test was used. To determine the correlation between human and KSHV viral gene expression, Spearman correlation analysis was used.An analysis of the sample size and statistical power for RNA sequencing was calculated using the R package, RnaSeqSampleSize. To achieve an 80% chance of detecting an effect, a sample size of 11, would be required when assuming an average of 2000 reads per gene, 16,000 total genes detected, a minimum fold change of 4, an FDR of 0.05, and 200 differentially expressed genes. For genomic-level expression analysis, adjusted p-values were calculated using the Benjamini and Hochberg method. To determine the gene expression difference between each KS and respective matched normal tissue sample from each participant, cellular genes with > log2 fold change of 2.0 with an adjusted p-value < 0.05 were identified. For smaller sets of comparisons, Student’s + T cell count of 38 cells/µL and HIV viral load (VL) of 443 copies/mL at the time of sample collection (Table 6 PBMCs and was higher for those with GI KS (1148 copies/106 PBMCs). Participants with skin KS had a lower median HIV VL (74 copies/mL) as compared to those with GI KS (5134 copies/mL). CD4+ T cell counts were similar in participants with skin KS (38 cells/µL) and GI KS (36 cells/µL).Nineteen participants contributed 22 KS samples with paired normal tissue, comprising 10 skin KS samples and 12 GI KS samples , encoding an angiogenic receptor VEGFR3, and Stanniocalcin 1 (STC1), a secreted glycoprotein that has altered expression in several malignancies. IL1A levels were increased in GI KS but repressed in skin KS  unique to skin KS and 58 DEGs unique to GI KS, and 26 common to both as compared to their respective normal tissues  genes were not significantly altered in expression when comparing KS lesions to their normal matched tissue samples , but not significantly altered in GI KS lesions as compared to matched normal tissues. Furthermore, increased expression of IL6 correlated with increased total KSHV gene expression in skin KS  but not in GI KS  was increased in both skin and GI KS  and CD274 (PD-L1) were modestly increased in skin KS, but not statistically significant in GI KS lesions.There was a wide range of expression changes in interferon-gamma (IFN-γ), which has been implicated to be a factor in combating KSHV infection . Only IFIL13, IL6, IL10, or IFNG expression levels by a diagnosis of KS alone as compared to KS with concurrent KAD in the skin or GI KS lesions , a proposed tumor suppressor, was increased in skin KS samples from those with KS alone whereas the gene expression was lower among those with KS with concurrent KAD  were separated by the presence of KS alone or KS with other KAD). There was no significant difference in We investigated whether canonical latent and lytic gene expression patterns were observed in KS biopsies. Among GI KS and skin KS, there was heterogeneity with respect to the KSHV expression patterns in the heatmap analyses Fig. A, B. In + T cell count of < 50 cells/µL. Patient A had no concurrent KSHV-associated diseases and had undetectable levels of circulating KSHV PBMC levels   were used to assess the impact of STC1 and FLT4 expression on viral infection and angiogenesis. Primary LECs were transfected with an siRNA control (siNTC) or siRNAs targeting try Fig. A based otry Fig. B. At 3-d25) Fig. C. The stA hallmark feature of endothelial cell infections with KSHV is tubule formation. To test the impact of STC1 and FLT4 on angiogenesis in LECs during KSHV infection, tubule formation was measured. As expected, the tubule formation, as measured by the total length branched structures, increased with KSHV infection at 3 dpi Fig. D. NotablKaposi sarcoma that manifests in the skin and GI tract leads to significant morbidity and mortality of individuals worldwide, especially among PWH. These analyses investigated similarities and differences in GI and skin KS lesions to discover host and viral factors associated with KS pathogenesis. To our knowledge, this is the first study evaluating and comparing KS lesions from different sites as well as the cellular and KSHV transcripts from KS tissues within a well annotated cohort from the United States. Heterogeneity was noted in the viral gene expression profile, but several canonical lytic genes were consistently detected irrespective of clinical characteristics. While we observed considerable heterogeneity in the expression of host cellular genes between GI and skin lesions as compared to their matched normal tissues, 26 common cellular genes of interest were identified between skin and GI lesions. These DEGs from KS lesions encode inflammatory cytokines as well as host factors associated with angiogenesis and immune regulation. Loss of function studies identified a role for STC1 and FLT4 in KSHV replication and virus-driven angiogenesis in KSHV-infected LECs.We noted highly complex patterns of KSHV gene expression in these KS lesions that do not fit the canonical classifications of the latent versus lytic gene expression program in KS. There was higher expression of ORF72/v-cyclin, compared to ORF73/LANA in both skin and GI KS lesions. This observation was consistent with a report from a previous study that analyzed KSHV RNA expression from KS tumors and suggests transcription initiation from an alternative promoter called LTd . The tra+ T cell count and HIV viremia was observed among those with GI KS. The heterogeneity between skin and GI samples may be related to the underlying clinical characteristics and concurrent KAD. Elevated inflammatory cytokines  are notably high in the circulation among individuals diagnosed with concurrent KSHV-associated diseases [Overall, the participants had a low median CD4diseases , 26, 27.diseases , 48.IFNAR2 alone was elevated in the skin KS lesions as compared to the normal skin. A wide range of IFNG expression changes were observed in both skin and GI KS lesions as compared to the normal tissue. Among these tissues that were evaluated, it is interesting to note that among the KSHV genes, the viral interferon regulatory factors (vIRF) expression, which is known to regulate antiviral response, was variable in its expression (highest expression of vIRF4) in all tissues [CCBE1 may indicate the diagnosis of KS alone and the absence of concurrent active KAD. As a possible tumor suppressor gene [CCBE1 in those with concurrent KAD and KS (where KSHV viremia in the circulation is expected to be higher) requires further evaluation.A previous study of 4 participants with HIV and cutaneous KS identified cellular genes associated with lipid and glucose metabolism ; however tissues , 50. In sor gene , the decBTAF, VMP1, DCHS1, VCAM1, PLXND1) . Additionally, the immune profiles seen in these analyses reinforce the use of chemotherapy sparing options, such as immunotherapy, which may increase T cell stimulation and reduce markers of T cell exhaustion associated with chronic HIV and KSHV infection [Among cellular genes in both GI and skin KS lesions as compared to their respective normal tissues, both FLT4 and STC1 were increased. FLT4 specifically has been noted in previous studies in HIV-associated KS , 44. Thenfection .The heterogenous clinical characteristics, such as the presence of concurrent KAD, prior treatment, KSHV levels and HIV characteristics, within this study are limitations that impact the generalizability of these findings. Despite these limitations, these findings provide a broad overview for subsequent analyses, specifically for GI KS that is associated with severe manifestations of this disease and remains rarely studied. Moreover, as there are no animal models for the study of KS, analyses of lesions from patients with KS provide important insights to factors and possible therapeutic targets that require further analyses.In summary, this study evaluates a selection of participants from the United States presenting with skin and GI KS. These analyses highlight differences in the KS immune profiles, angiogenic factors and KSHV gene expression by site of involvement. Future studies may include investigating the spatial association of these gene expression patterns across numerous samples to better understand how KSHV-infected cells interact with the immune system and the tumor microenvironment to impact these changes.Additional file 1: Table S1. KSHV inflammatory cytokine syndrome case definition from Polizzotto MN, Uldrick TS, Wyvill KM, Aleman K, Marshall V, Wang V, et al. Clinical Features and Outcomes of Patients With Symptomatic Kaposi Sarcoma Herpesvirus (KSHV)-associated Inflammation: Prospective Characterization of KSHV Inflammatory Cytokine Syndrome (KICS). Clin Infect Dis. 2016;62(6):730-8. Tables S2 and S3. Pathway enrichment for differentially expressed genes (DEGs) in skin (S2) and GI (S3) samples. Table S4. Spearman correlation analysis between specific human gene expression and total KSHV gene expression. Figure S1. After RNA sequencing, gene expression profiles of combined human and viral genes were analyzed using principal component analysis for skin samples (A), GI samples (B), and combined (C). “N” at the end of sample names are the normal tissue samples. “T” in sample names refers KS tumor samples. D. KSHV transcript per million (TPM) values were combined for all KSHV genes. Stronger red backgrounds indicate higher KSHV expression. Figure S2. Human gene expression patterns in matched samples. A. Inflammatory and interferon genes associated with viral pathogenesis or immune responses are plotted. B. T cell genes associated with viral pathogenesis or immune responses are plotted. Asterisks (blue* for GI and orange * for skin) represent statistically significant  genes, in individual GI  and skin  samples. Figure S3. Induction of STC1 during infection and depletion with siRNAs. Induction of STC1 secreted protein (A) and transcript (B) for de novo infected HDLEC cells and corresponding reduction with siSTC1 in tube formation assay. Similarly, FLT4 transcript was depleted with siFLT4 (C). Samples shown as paired to respective controls . RT-qPCR was used to determine expression differences. D. Western blotting for STC1 and FLT4 (E) after KSHV infection and transfection with siRNAs. Figure S4. CRISPR screens previously reported and skin KS expression changes . Depleted targets (blue dots) were hits from CRISPR screens for killing KSHV-infected cells in PEL cells (A) or endothelial cells (B). Red dots depict the average fold changes in RNA expression from skin KS lesions compared to normal skin (introduced in Fig.


In the current study, apple-pectin-based novel nanofibers were fabricated by electrospinning. Polyvinyl alcohol (PVA) and apple pectin (PEC) solution were mixed to obtain an optimized ratio for the preparation of electrospun nanofibers. The obtained nanofibers were characterized for their physiochemical, mechanical and thermal properties. The nanofibers were characterized using scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy and thermogravimetric analysis (TGA). Furthermore, an assay of the in vitro viability of free and encapsulated probiotics was carried out under simulated gastrointestinal conditions. The results of TGA revealed that the PVA/PEC nanofibers had good thermal stability. The probiotics encapsulated by electrospinning showed a high survival rate as compared to free cells under simulated gastrointestinal conditions. Furthermore, encapsulated probiotics and free cells showed a 3 log (cfu/mL) and 10 log (cfu/mL) reduction, respectively, from 30 to 120 min of simulated digestion. These findings indicate that the PVA/PEC-based nanofibers have good barrier properties and could potentially be used for the improved viability of probiotics under simulated gastrointestinal conditions and in the development of functional foods. Lactobacillus acidophilus, have gained significant attention in recent years due to their potential health benefits. These beneficial bacteria have been associated with improving gut health, enhancing the immune system and even aiding in weight management. However, the viability and stability of probiotics during storage and digestion pose significant challenges [Probiotics, particularly allenges . To overallenges .L. acidophilus.Electrospinning has emerged as a promising method for the production of nanofibers with high surface-to-volume ratios, uniform structures and excellent mechanical properties. By incorporating probiotics within electrospun nanofibers, their viability and stability can be significantly improved . In thisPectin has received a lot of attention as a desired biopolymer, with apples being one of its main sources. It is a complex polysaccharide made up of galacturonic acid and other natural sugar residues. Apple pectin, in particular, has demonstrated excellent biocompatibility and biodegradability, making it suitable for food packaging and energy storage . AdditioThe use of apple-based pectin in electrospinning processes offers several advantages. Firstly, pectin can form homogenized solutions, which provide a suitable environment for probiotic survival and growth. The hydrophilic nature of pectin ensures the retention of moisture, thereby preventing the dehydration of the probiotic cells . MoreoveL. acidophilus) by adopting the novel approach of electrospinning. In this study, nanofibers of apple pectin and PVA were fabricated and further characterized for physiochemical, structural and morphological properties. Moreover, the viability of free and encapsulated probiotic cells was assessed under thermal and simulated gastrointestinal conditions. This study will provide meaningful information about the survival of probiotics and open a new window for the development of new functional foods and drug delivery.This study aims to assess the viability of a probiotic strain  agar used in this study were purchased from Sigma-Aldrich Shanghai Co., Ltd., Shanghai, China. Other chemicals and reagents used in this study were used without further purification.Pectin  was obtained from Sigma-Aldrich, Shanghai, China. The polyvinyl alcohol , L. acidophilus was prepared by following the method of Aider-Kaci et al. [10 cfu/mL.A bacterial culture of i et al. , whereasw/v) PVA solution was prepared by adding 10 g of PVA to 100 mL of distilled water on a magnetic stirring plate  at 90 °C for 2 h. Next, 10% (w/v) pectin (PEC) solution was prepared by adding 10 g of PEC to 100 mL of distilled water on a magnetic stirring plate at 80 °C for 3 h. A standard ratio of PVA/PEC blends was formulated as by Xu et al. [For the fabrication of nanofibers, 10% . The carriage of the nanospider was filled with PEC/PVA solution and a conductive sheet was applied for the collection of flying nanofibers. The conduction rate was set at 140 mm/s, the voltage was set at 118.8 kV and a distance of 10 cm was maintained between the carriage and collector sheet. The process of fabrication was started and the solution was completely consumed after a few hours. The foil was taken out of the machine and a sheet of nanofibers was obtained upon the collector fabric.The bacterial suspension containing L. acidophilus, was determined by following the method of Xu et al. [The encapsulation efficiency (%) of the PEC/PVA nanofibers loaded with probiotics, u et al. . BrieflyZeta potential is an important factor to determine the colloidal dispersion of nanofibers. The zeta potential of the PEC/PVA nanofibers was determined by the method of Liu et al. , via ZetAn electronic digital micrometer  was used to determine the thickness (nanometers) of the PEC/PVA nanofibers. The thickness was determined at various positions of the nanofibers. An average of ten replicates were taken for the mean values.The fabricated nanofibers of PEC/PVA were assessed for their barrier properties, such as thickness, tensile strength (TS) and elongation at break (EAB). The thickness of the nanofibers was determined using scanning electron microscopy, whereas to assess the tensile strength and EAB, the nanofibers were subjected to control stretching force until they broke. The applied force and resulting deformation were measured to calculate the TS and EAB using the following formulas:The blank and probiotic-loaded nanofiber mats were subjected to scanning electron microscopy, SEM , for the morphological characterization. The diameters (±SD) of the PEC/PVA nanofiber (blank and probiotic-loaded) mats were measured at 100 different positions on an SEM image.−1 at room temperature.Fourier transform infrared spectroscopy  was performed for the molecular characterization of the free and probiotic-loaded PEC/PVA nanofibers. The secondary structure was assessed using OMNIC Software 8.3  by removing background peaks. A total of 32 scans were performed between 400 and 4000 cm0) and probiotic-loaded (P1), a thermogravimetric analyzer, TGA , was used.To analyze the thermal behavior of the PVA powder, PEC powder and PVA/PEC nanofibers, both blank  conditions, the method described by Singh et al.  was follp < 0.05.The data were statistically analyzed with an appropriate model of SPSS for Windows . The collected data were statistically analyzed through mean values and standard deviation in order to evaluate the statistical significance of every parameter . The sigEncapsulation efficiency is a critical factor in probiotic encapsulation as it influences the probiotic’s protection, viability, stability, controlled release and dose consistency. The overall quality, effectiveness and reliability of a probiotic can be enhanced by optimizing encapsulation efficiency . The res0) and probiotic-loaded (P1) PEC/PVA nanofibers have been listed in 0) and probiotic-loaded (P1) nanofibers showed negative values. It has been stated by Coelho Braga de Carvalho et al. [0 and P1 nanofibers, respectively. The mean results show significant (p < 0.05) differences for zeta potential. The current findings reveal that the PEC/PVA nanofibers loaded with probiotics (P1) showed lower zeta potential compared to the blank PEC/PVA nanofibers (P0). Thus, the decrease in zeta potential from −7.55 mV (P0) to −10.22 mV (P1) indicated that the probiotics were successfully encapsulated within the nanofibers of PEC/PVA. Ceylan et al. [Zeta potential represents the electrical potential at the interface between a particle or a surface and the surrounding liquid medium. It quantifies the magnitude and polarity of the charge present on the surface of the particle or material. Zeta potential is an important parameter to determine in nanofibers because it provides insights into their surface charge and stability . Nanofibo et al.  that zetn et al.  also obs0) and probiotic-loaded (P1) PEC/PVA nanofibers. Determining the moisture content in nanofibers is important for maintaining fiber stability, optimizing processing conditions, controlling material performance and ensuring storage stability [0) was observed to be 14.11%, whereas the moisture content was increased in the probiotic-loaded PEC/PVA nanofibers (P1), at 14.27%. The increased moisture content observed in the probiotic-encapsulated nanofibers in the current study can be attributed to several factors such as the fact that the encapsulation matrix, pectin and PVA have efficient water retention properties, which may increase surface area and reduce moisture loss. These factors collectively contribute to the higher moisture content, which can be beneficial for the viability and functionality of the encapsulated probiotic cells [Moisture analysis was conducted for both the blank  and probiotic-loaded (P1), are listed in p < 0.05) differences for both blank and probiotic-loaded nanofibers. The average thickness for P0 (blank) was recorded as 0.127 mm, whereas for P1 (probiotic-loaded), the average thickness was recorded as 0.137 mm. A slight increase in the thickness of nanofibers has been affiliated with the probiotic encapsulation, as Ajalloueian et al. [The thickness of nanofibers is a vital as it directly impacts the performance, functionality, mechanical properties, transport phenomena, fabrication process and application-specific requirements of nanofiber-based materials . The resn et al.  reported0) and probiotic-loaded (P1) PEC/PVA nanofibers in terms of tensile strength (TS) and elongation at break (%) were determined. Tensile strength refers to the maximum stress a material can withstand before it breaks or fails under tension. It is a measure of the material’s resistance to being pulled apart, whereas elongation at break (EAB) is a measure of the material’s ability to stretch or deform before it eventually fractures. It indicates the ductility or flexibility of a material. Nanofibers with high elongation at break can undergo substantial deformation without breaking, making them suitable for applications where flexibility and resilience are required [p < 0.05) difference. These results indicate that the tensile strength of the nanofibers significantly increased after the encapsulation of probiotics. Likewise, the mean results for EAB show a significant increase from the blank to probiotic-loaded nanofibers from 22.05% to 36.98%. The mechanical properties of the nanofibers increased after the encapsulation of probiotics. This result is supported by the findings of Duman & Karadag [The barrier properties of both the blank  and probiotic-loaded (P1). In the PVA powder, the peak at 712 cm−1 indicates the presence of CH2 rocking vibrations, which are mainly characteristic of PVA. Pectin is a complex polysaccharide, and its FTIR spectrum exhibits various peaks related to different functional groups. The peak at 850 cm−1 is attributed to C-C stretching vibrations or the presence of PVA side chains. The peaks at 1000 cm−1 and 1078 cm−1 correspond to C-O stretching vibrations, which are typical for PVA as well as pectin. The peak at 1250 cm−1 indicates the presence of C-O-C asymmetric stretching vibrations in PVA. The peak at 1617 cm−1 is attribute to the presence of C=O stretching vibrations in PVA, suggesting the presence of carbonyl groups, whereas in the PEC powder, the peak at 1617 cm−1 indicates the presence of carboxylate groups (-COO-), which are characteristic of pectin. The peaks at 1878 cm−1 and 2989 cm−1 indicate O-H stretching vibrations, which are characteristic of PEC. The peak at 3250 cm−1 corresponds to O-H stretching vibrations, suggesting the presence of hydroxyl groups in PVA, and the peak at 3250 cm−1 corresponds to O-H stretching vibrations, indicating the presence of hydroxyl groups in pectin. The peaks observed for PVA and pectin (P0) in the individual spectra are expected to appear in the combined solution as well. The relative intensities of these peaks depend on the concentration and ratio of PVA and pectin in the solution. However, in the probiotic-loaded nanofibers (P1), the presence of probiotics in the combined solution introduced additional peaks and modified the intensities of existing peaks in the spectrum. Specific changes can be observed in the spectra, which indicates the interaction between the probiotics and the PVA–pectin matrix. These changes are related to new functional groups or alterations in the molecular structure of the components. Various studies have observed similar alterations of peaks that indicated the interaction of compounds with each other [ch other .The thermal stability of the PEC/PVA nanofibers was assessed in the current study, and the results are shown in 1) are shown in 1) was recorded as 9.33 log (cfu/mL) at 30 min, which was later reduced to 7.35 log (cfu/mL) at 120 min. These findings reveal that the viability increased from 0 log (FCs) to 7.35 log (cfu/mL) (P1) among free cells and encapsulated cells at 120 min, respectively. The results of the current study are supported by Atraki & Azizkhani [The human stomach is a highly acidic environment with a low pH that can range from 1 to 3, which poses a significant challenge to the survival of probiotics, as it can lead to a decrease in their viability . By subj1) were subjected to simulated intestinal conditions (SICs). The results are presented in 1) were more viable than free cells, with a maximum viability of 9.31 at 30 min and a minimum viability of 7.54 at 120 min, though cells were still viable. The findings in Similarly, both free cells (FCs) and cells encapsulated in nanofibers  improves the barrier properties of nanofibers. However, equal ratios of pectin and PVA (10:10) demonstrate excellent spinnability in electrospinning. This combination proves to be a promising approach for increasing the viability and stability under stressed conditions. However, future work could investigate the modification of pectin by means of esterification or using various types of pectin with diverse molecular weights and average chain lengths, which may enhance the survivability of probiotics. The development of new nanofibers opens a new window for the increased survival of probiotics, which may be helpful for the development of new functional foods.


Vibrio vulnificus infections have emerged as an increasingly serious public health threat along the German Baltic coast. To manage related risks, near real-time (NRT) modelling of V. vulnificus quantities has often been proposed. Such models require spatially explicit input data, for example, from remote sensing or numerical model products. We tested if data from a hydrodynamic, a meteorological, and a biogeochemical model are suitable as input for an NRT model system by coupling it with field samples and assessing the models’ ability to capture known ecological parameters of V. vulnificus. We also identify the most important predictors for V. vulnificus in the Baltic Sea by leveraging the St. Nicolas House Analysis. Using a 27-year time series of sea surface temperature, we have investigated trends of V. vulnificus season length, which pinpoint hotspots mainly in the east of our study region. Our results underline the importance of water temperature and salinity on V. vulnificus abundance but also highlight the potential of air temperature, oxygen, and precipitation to serve as predictors in a statistical model, albeit their relationship with V. vulnificus may not be causal. The evaluated models cannot be used in an NRT model system due to data availability constraints, but promising alternatives are presented. The results provide a valuable basis for a future NRT model for V. vulnificus in the Baltic Sea.Over the last two decades,  Vibrio spp. are ubiquitous members of the ocean and freshwater microbial communities. More than 100 different Vibrio species have been identified [Vibrio spp. play an important role in marine nutrient cycles [Aliivibrio fischeri and squids) [Vibrio spp. are human pathogens [Vibrio cholerae (O1 and O139) are the causative agents of the intestinal disease cholera [V. cholera strains and other Vibrio spp. cause vibriosis, with symptoms ranging from gastroenteritis to skin and soft tissue infections with necrotizing fasciitis, septicaemia, or even fatal septic multi-organ failure [entified ,3. Vibrit cycles ,5, and s squids) . About aathogens ,8. For e failure ,9. InfecV. alginolyticus, V. cholerae (non-O1/non-O139), V. parahaemolyticus, and V. vulnificus [V. vulnificus are often severe  and have high mortality rates [V. vulnificus, is a public health concern [Vibriosis outbreaks in Northern Europe are often attributed to lnificus ,12,13. T concern ,15,16.Vibrio, which causes vibriosis, particularly of V. vulnificus, and predicting their abundance is an important component for the effective management of Vibrio-related public health risks [Vibrio vulnificus is known to form short but intense blooms under favorable conditions [Understanding the ecology of th risks . With grnditions ,19. It hnditions ,21,22,23V. vulnificus quantification from water samples at seven beaches in the federal state of Mecklenburg–Western Pomerania (MV) during the summer months, and awareness campaigns for visitors [The Baltic Sea has low salinity and one of the highest warming rates in marine ecosystems worldwide ; thus, ivisitors . In the visitors .Vibrio abundances and infection risks in near real-time (NRT) [Vibrio Map Viewer provided by the European Centre for Disease Prevention and Control . It provides a species-independent relative Vibrio infection risk index. The index is calculated based on sea surface temperature (SST) and sea surface salinity (SSS) from remote sensing products, as well as ocean reanalysis and forecast products [Vibrio spp. show different responses to the same environmental conditions [Vibrio occurrence probabilities or quantities of a certain species may provide more robust results for NRT modelling.Advances in remote sensing and model products provide opportunities for modelling of me (NRT) . One exaproducts . Howevernditions ,21,28,29nditions ,13,17,30V. vulnificus quantities in the Baltic Sea would be a step towards better management of the increasing risks of Vibrio-related public health issues in the future [V. vulnificus by coupling the model data with more than 600 samples of V. vulnificus. Moreover, we identify parameters particularly suited for predicting V. vulnificus abundances in the western Baltic Sea by applying a novel network detection method, the St. Nicolas House Analysis [V. vulnificus and offer suggestions for improvements. Moreover, using a time series of 27 years of modelled daily SST, based on validated hindcast simulations, we attempt to detect the footprint of climate change on V. vulnificus season length and detect hotspots of frequent V. vulnificus occurrence along the German Baltic coast.Despite suggestions that creating an operational NRT modelling system for e future ,17,22,27Analysis . We discV. vulnificus in the south-western Baltic Sea. A list of abbreviations and acronyms is provided at the end of the article.The following section outlines the study area, materials, and methodology used to identify the environmental drivers of The German coast of the Baltic Sea is characterised by large bights, the “Schlei” (a narrow inlet in the north-west of the study region of ∼30 km length), fjords, and the shallow “Bodden” in the east . The latV. vulnificus [SST shows strong seasonal dynamics. During the summer months, SST reaches averages of 17 °C in the open sea and up to 20 °C in sheltered and shallow areas. Heatwaves may cause positive SST anomalies of up to 5 °C, as observed in 2018 ,22. The lnificus ,20. Morelnificus .Vibrio datasets used in this study were provided by the Ministry of Social Affairs, Youth, Family, Senior Citizens, Integration and Equality Schleswig-Holstein (SoZMI), and the State Agency for Health and Social Affairs Mecklenburg–Western Pomerania (LAGuS). The former dataset consists of samples taken along the Baltic coast of the German state of Schleswig-Holstein (SH) between 2014 and 2021. The latter provides measurements from Mecklenburg–Western Pomerania (MV) between 2008 and 2021. Samples were taken during several independent monitoring projects of the federal states, which used different sampling periods and cultivation-based identification and quantification of Vibrio spp., as described in the following. The SH dataset contains samples from a campaign in 2014 and incident-based samples from 2015 to 2021. Incident-based samples were taken after reported cases of Vibrio infections. For Vibrio quantification and species identification, 250 mL of water was sampled from bathing areas at 30 cm below the surface according to SH bathing water directive BadGewQualV SH [TM Vibrio  and incubated for 24 h at 36 °C and 5% COTM Marine Broth with 1.5% Agar–Agar, both BD Diagnostics, Franklin Lakes, NJ, USA) for a further 24 h. The colonies of Vibrio species  were counted, and colony-forming units (CFUs) extrapolated for 1 ml seawater (CFU/mL). Vibrio identity was verified using individual colonies and a MALDI-TOF analysis (see [Vibrio). In MV, water samples were taken every 2 weeks at 7 stations starting in 2008. Sampling began each summer after a threshold water temperature of 17–18 °C was reached [Vibrio identification and quantification in water samples was done using a cultivation-based approach, similar to the SH dataset. For a description of the cultivation, refer to [V. vulnificus abundance in powers of 10 (the most probable number technique). This entailed screening Vibrio growth along a dilution series and recording the highest dilution with visible colonies. The experiments were conducted in triplicate. To make the SH and MV datasets comparable, the SH records were converted into corresponding powers of ten.The QualV SH . Moreovesis  ,42 was uce Model ,45. Bounce Model  for detahttps://www.bokniseck.de/, accessed on: 27 November 2022) [r), mean absolute error (MAE), and bias between modelled and observed data.Model results were validated with in situ data from three in situ sampling stations . At Bokner 2022) . The Leier 2022) , daily aData on nutrients, dissolved oxygen (O biomass ) were ta biomass . The rea biomass ,50, coup biomass ,52. The  biomass . The modV. vulnificus sampling program in 2008.We accessed the dataset through CMEMS OPeNDAP Python API and downloaded all daily averages of the surface layer in our study region since the beginning of the The COSMO-REA6 dataset is a reanalysis product from the German Weather Service DWD . It is bhttps://opendata.dwd.de/climate_environment/REA/, accessed on: 6 December 2022).We downloaded daily averages of surface air temperature (SAT), total precipitation, solar irradiation, and wind speed  for the study period (2008–2019) from the open data server of the DWD , valid data was not always available directly at the coastal sampling locations. In such situations, all data within a predefined radius around the sampling location were averaged. The search distance was increased iteratively until valid predictions were found or a maximum radius of 1.5 × the grid cell resolution was reached.Several studies indicate a lead time between some environmental parameters  and bundance ,38,54. FV. vulnificus quantities, and plotted in correlation matrices. Spearman’s rank correlation was used to account for potential non-linearity of underlying processes. The window with the highest correlation coefficient was generally used as the time lag in further analysis.The 30-day time series were analysed using a moving window of flexible size . For eacV. vulnificus quantities not only depend on absolute values of a parameter, but also on the duration of an event [It has already been observed that an event ,55. TherV. vulnificus enters a viable but non-culturable state (VBNC), which is a survival strategy altering their metabolism if water temperatures drop below ∼13–15 °C [Previous studies in temperate regions have shown that 13–15 °C . For resmean12–15. This stands for the average SST between 12 and 15 days before Vibrio sampling.In the following, lag windows of variables will be denoted as, for instance, SSTV. vulnificus by calculating Spearman’s rank correlation coefficient . SNHA provides an approach for initial data analysis by visualizing associations between variables in non-linear multivariate data [Initially, we tested the ability of our models to reproduce known ecological characteristics of ate data . It rankate data . In contate data ,57.Recently, Hake et al.  proposedV. vulnificus samples and environmental parameters to unveil a network between the different parameters. We used the Python implementation SNHA4py  [SNHA was applied to the complete dataset of 0230103)  and the 0230103) ) suggestV. vulnificus quantity. Our high-resolution SST product provides the unprecedented opportunity to identify hotspots with frequently favourable SST and regions with high warming rates since 1995. To identify these regions, we first calculated the V. vulnificus occurrence probability with logistic regression on our V. vulnificus samples and corresponding modelled SST values from our GETM realisation. Next, we counted the days in each year where SST exceeded the temperature of the 33% probability of V. vulnificus occurrence . Trends were estimated with Sen’s slope, and their statistical significance was ensured with the Mann–Kendall test using the pyMannKendall package [Preliminary results identified SST as the most important parameter determining  package .r of 0.99 and 0.97 for SST and SSS, respectively). However, the model appears to slightly underestimate SST . For SSS, the MAE is approximately 0.4 PSU and the bias is negligible (−0.03 PSU), although SSS at Boknis Eck appears to be moderately overestimated (bias of 1.88 PSU).Validation of the high-resolution hydrodynamic model demonstrates the accuracy of both SST and SSS products . Both paV. vulnificus samples (408 in MV and 213 in SH). For each environmental parameter, we created correlation matrices with V. vulnificus quantities. In most cases, the lag window with the highest Data of all environmental parameters were available for a total of 621 V. vulnificus quantities and all environmental parameters , correlate significantly with V. vulnificus quantity (p > 0.05), albeit most mean0–11, SATmean0–16, Omean0–02 and SSSmean0–6 .mean0–11) forms the centre of the network and is connected to 10 other parameters, including biogeochemical, meteorological, and physical parameters, as well as the V. vulnificus quantity. The biogeochemical parameters sharing an edge with SST are mean oxygen (Omean0–0), mean nitrate (NOmean7–25), mean ammonium (NHtrend9–10), and the oxygen trend (Otrend5–16). The meteorological parameters include mean surface air temperature (SATmean0–16), wind speed (WSmean2–17), and the trend of surface air temperature (SATtrend7–29). The physical parameters connected to the mean sea surface temperature are the sea surface temperature 180 days before sampling (SSTtrend11–26). With an mean0–11) and surface air temperature (SATmean0–16) are closely related.We applied SNHA to derive a network between all parameters . The meaV. vulnificus were inferred from seven parameters. Among them are mean sea surface temperature , phosphate (POmean22–224), nitrate (NOmean7–253), and chlorophyll (Chlmean6–15) have a link to mean sea surface salinity (SSSmean0–6), while mean nitrate (NOmean7–253) and the trend of ammonium (NHtrend9–104) are connected to the average sea surface temperature SSTmean0–11. These nutrients are, thus, indirectly connected to the V. vulnificus quantity.Direct edges to V. vulnificus season, we calculated the occurrence probability based on SST with logistic regression. The fit reveals a slow increase of occurrence probability up to ∼17 °C. At 17.6 °C, the probability reaches 33% and increases sharply by about 20% per degree  for 1995 (a) and 2021 (b). Apart from the trend towards a longer season in 2021, the season length exhibits similar geographic patterns in both years. Longer than average seasons occurred, for example, in the inner Flensburg Fjord in the north-west of the study region, the Bay of Lübeck in the centre, and the easternmost areas of the study region  modelling system for the German Baltic Sea by combining more than 600 V. vulnificus abundance in regions with a temperature-driven seasonal cycle, e.g., [V. vulnificus occurrence increased rapidly once SST exceeded ∼17 °C. The highest numbers of V. vulnificus CFUs were observed when SST was above 20 °C. These dynamics agree well with reports from other studies, e.g., [Our results largely confirm the important constraining influence of SST on e, e.g., ,61,62. Is, e.g., ,22,63.V. vulnificus can persist at lower temperatures before entering the VBNC state. Oliver et al. [V. vulnificus towards lower water temperatures in autumn before entering the VBNC state, we included SST 180 days before sample acquisition  and in r et al.  and 13 °r et al. ). The 14V. vulnificus quantity and SATmean0–16. Given the strong correlation between SATmean0–16 and SSTmean0–11  differ from findings in other regions, such as e.g., the North Sea [V. vulnificus. Hence, precipitation appears to be more important in regions where high SSS limits V. vulnificus growth. In the brackish Baltic Sea, precipitation may simply reflect variations of the weather in the days and weeks before the sampling. Further, the GETM hydrodynamic model takes the effect of precipitation on SSS into account. This means that potential effects of precipitation on salinity are already included in the SSS parameter.Weak negative correlations between orth Sea , or Hawaorth Sea . In all V. vulnificus and different nutrient concentrations exist . These alternatives have only become available in recent years and do not cover the entire period of Vibrio monitoring. Therefore, thorough model comparisons would be required if model training and prediction are carried out on different datasets.Reanalysis products were used for meteorological and biogeochemical parameters. Both reanalyses were chosen based on their temporal coverage, which overlaps with most of the om CMEMS , the opeom CMEMS , and theV. vulnificus quantities through cultivation and CFU count/ml, differences in experimental approaches between laboratories exist. The datasets were homogenised, but this came at the expense of losing the continuous scale of the data from SH in favour of the less detailed categorical system from MV. Moreover, existing sampling strategies primarily monitor Vibrio quantities during conditions favourable for V. vulnificus or after reported infections. Consequently, July and August contribute to over 70% of all the samples. The accuracy of a future iteration of the NRT model would certainly benefit from additional samples derived from consistent, year-round sampling campaigns. Such sampling efforts would also provide the opportunity to extend our approach to other  pathogenic Vibrio species, such as V. parahaemolyticus, V. cholerae or V. fluvialis, which have been studied less intensely in the German Baltic Sea [It is crucial to keep in mind that the quality of the underlying data limits the effectiveness of any statistical model. In this study, we were able to generate a dataset with more than 600 samples, although these samples were acquired in two different federal states with slightly different sampling protocols. While both states assessed ltic Sea ,74.Vibrio data to train the NRT models, an additional resource may be found in the DNA databases, such as the NCBI Sequence Read Archive . These databases contain large amounts of marker-gene or metagenomic sequencing data from around the world, and could be compiled to search for relative abundances (proportions) of Vibrio amongst microbial communities. At the very least, the data could provide presence/absence-based information for different sampling locations around the globe. This approach would, however, require considerable computational efforts, as well as close curating of the datasets, which are beyond the scope of this study.In another attempt to gain additional V. vulnificus and their distribution have been widely discussed and can already be observed. For instance, higher numbers of vibriosis cases are reported during exceptionally warm summers [V. vulnificus is likely to occur has been extended almost throughout the entire southwestern Baltic Sea since 1995, due to the rising water temperatures. In some areas, the season has extended at rates exceeding one day per year. These trends, however, are significant only in areas that experience the highest rates of warming. These mainly include sheltered and shallow bays, as well as the area east of the island of Usedom. East of Usedom, the water is relatively turbid, leading to more intense heating of the water [V. vulnificus season length, defined as days with SST exceeding 17 °C. In other regions, trends in season length remain insignificant, likely due to the underlying climate variability obscuring them.Globally, the Baltic Sea is the marine region with the highest rate of warming . Along t summers ,13. The he water ,77. In she water , therebyhe water  projecteV. vulnificus occurrence probability of 33% . When we used higher probabilities as the threshold , trends remained insignificant in almost the entire study area. We suspect that this was due to an increase in variance as the temperature threshold rose, likely due to an increased impact of extreme events. Under such conditions, the Mann–Kendall test may be unable to identify comparably small slopes [V. vulnificus season length requires further improvement, the influence of climate change on the occurrence of V. vulnificus was evident from our data. As the season is closely linked to water temperature and, therefore, the public bathing season, an increase in human exposure to V. vulnificus can be expected [The influence of climate variability also explains our rather arbitrary choice for the SST threshold at a l slopes . While tV. vulnificus season may also lead to increased stress on densely colonised aqua- and larvicultures by members of the V. vulnificus, which primarily infect marine hosts. While this can cause mass mortalities within the aquacultures themselves, derived seafood can provide an additional source for human contact and subsequent vibriosis [V. vulnificus, but also includes other zoonotic Vibrio members, such as V. parahaemolyticus [V. alginolyticus [Vibrio species and/or serotypes.Increased SST and a longer ibriosis ,80. Infeolyticus  or (rareolyticus . It may,V. vulnificus quantities in the German Baltic Sea. The most significant factors were SST, SAT, SSS, dissolved oxygen, and precipitation. This is the first step in creating an NRT model to forecast the occurrence and quantities of V. vulnificus. Given that climate change is increasing human exposure to V. vulnificus, such an NRT prediction system is crucial for managing future infection risks. To develop a reliable NRT model, we advise standardising Vibrio spp. sampling strategies and measurement methods across all federal agencies.By analysing environmental data from models and reanalyses, we found key ecological factors for predicting