A. baumannii infections. Peptide vaccines offer an attractive and promising preventive strategy against A. baumannii.The lack of appropriate vaccines is an obstacle to the effective management of A. baumannii outer membrane protein K (OMPK) using comprehensive bioinformatics and detailed molecular docking analysis.In this study, we identified specific T cell epitopes of A. baumannii OMPK were predicted by three tools namely IEDB, SYFPEITHI, and ProPred. The predicted epitopes were shortlisted based on several analyses including prediction scoring, clustering, exclusion of human similarity, considering immunogenicity and cytokine production, and removal of toxic and/or allergen epitopes. The epitopic peptides with high prediction scores and appropriate properties containing both class-I and class-II T cell epitopes were selected. Two of these class I/II epitopic peptides were chosen for molecular docking studies and assessing their physicochemical properties as vaccine candidates.Both class-I and class-II T cell epitopes of The results showed many T-cell epitopes of OMPK that could be evaluated for possible immunogenicity. Two of these epitopes (containing both class-I and II epitopes) had high prediction scores, were predicted by several tools, attached to several HLAs, and had the best docking score. They had different physicochemical properties and were conserved among Acinetobacter species.A. baumannii OMPK high immunogenic class-I and class-II T cell epitopes and introduced two promising high immunogenic peptides as vaccine candidates. It is recommended to perform in vitro/in vivo investigation of these peptides to determine their true efficacy and efficiency.We identified the The online version contains supplementary material available at 10.1186/s13104-023-06409-9. Acinetobacter baumannii, an opportunistic pathogen responsible for hospital-acquired infections, is considered a serious threat to public health [A. baumannii is responsible for approximately 20.9% of all hospital-acquired infections in Europe, the Eastern Mediterranean, and Africa [A. baumannii infections and occurs mostly in patients receiving mechanical ventilation in ICUs [A. baumannii was present in 79.9% of all cases of acquired pneumonia and ventilator-associated pneumonia (VAP) [Bacteria of the genus Acinetobacter are non-motile Gram-negative coccobacilli that are ubiquitous in nature but are mainly found in soil, water, and sewage . Acinetoc health . This bac health . In inted Africa . Hospita in ICUs . A recenia (VAP) .Acinetobacter baumannii can be spread through respiratory droplets such as respiratory secretions, sneezes, and saliva, as well as through person-to-person contact, skin picking, and contact with contaminated surfaces [A. baumannii ability to propagate easily is mainly due to three characteristics: desiccation resistance, the ability to form biofilms on nonliving surfaces, and a propensity to adhere to host cells [A. baumannii has made it difficult to control using common antibiotics. Therefore, the development of effective vaccines is an alternative means to prevent infections caused by A. baumannii [A. baumannii as a key priority for the development of new medical countermeasures, including vaccine development [surfaces . A. baumst cells . The incaumannii . As a reelopment .A. baumannii [A. baumannii infection. However, none of them have yet received approval for use in humans, and further research is still needed to develop a suitable vaccine candidate against A. baumannii [Over the past decade, research efforts have identified several promising immunomodulatory strategies that could lead to a safe and effective vaccine against aumannii . Severalaumannii .A. baumannii [A. baumannii is the presence of a dense polysaccharide capsule that shields most of the outer membrane antigens from immune recognition [A. baumannii vaccines are often composed of antigens present on the bacterial outer membrane, such as OMPA, OMP22, and OMPK [Outer membrane proteinsaumannii . One of ognition . OMPs arognition , 14. Theand OMPK .A. baumannii OMP that is specific for the nucleoside-forming ion channels. This protein is equivalent to OMPK in the fish pathogen Vibrio harveyi [A. baumannii OMPK has significant immunogenic properties and is considered a promising vaccine candidate [A. baumannii, although no accurate information about OMPK epitopes vaccine potential can be found in the literature.Outer membrane protein K harveyi . Previouandidate –17. BothA. baumannii-related diseases. Today, in addition to molecular approaches, computer calculations and bioinformatics studies have provided a new method for vaccine creation through in silico epitope prediction. Numerous studies, similar to the present study, have utilized bioinformatics and sometimes laboratory analysis to investigate suitable vaccine candidates for various bacteria [A. baumannii OmpK through comprehensive bioinformatics and detailed molecular docking analysis, based on cell-dependent immune responses. In addition, this study introduces two T-cell epitopic peptides derived from OMPK, which exhibit suitable immunological and physicochemical properties, as potential A. baumannii vaccine candidates.The development of epitopic (peptide) vaccines could offer an attractive and viable treatment option for bacteria –23. The A. baumannii OMPK was retrieved from the protein database of NCBI .The sequence of http://www.allelefrequencies.net/), the prediction servers, or using our previous experiences on K. pneumoniae antigens [The most frequent human leukocyte antigenantigens –26 (Addihttp://www.syfpeithi.de/bin/MHCServer.dll/EpitopePrediction.htm), IEDB (http://tools.iedb.org), and ProPred (http://crdd.osdd.net/raghava/propred/). For the prediction of class-I and class-II epitopes, the percentile ranks of ≤ 1 and ≤ 10, respectively, were selected as the thresholds in IEDB. In ProPred-I and SYFPEITHI servers, the score of ≥ 10 was used as the threshold, whereas in ProPred-II all the predicted epitopes were chosen. Epitopes of 9 and 15 residues were chosen for the prediction of class-I and class-II epitopes, respectively.Similar to our previous studies , 27, thehttp://tools.iedb.org/cluster/), and an epitope of each cluster was selected for advanced analyses based on its higher prediction score compared to the others. The immunogenicity of class-I epitopes was predicted by a specific IEDB tool (http://tools.iedb.org/immunogenicity/). The potency of class-II epitopes in interferon-gammahttp://crdd.osdd.net/raghava/ifnepitope/).Regarding ≥ 70 similarities, the predicted epitopes were clustered with the IEDB clustering tool . The percentage of human similarity was estimated by two indicators of coverage and identity in the BLASTP server using the human proteome (taxid 9606) as the reference organism. The ≥ 90% similar epitopes to human proteome were excluded from further analyses. The AllerCatPro [The T cell epitopes were assessed for possible toxicity using the ToxinPred server .Epitopes were searched for any experimental record using the IEDB home page  used as a template.The structure of the OMPK protein was generated using the SWISS-MODEL server , with tThe PEP-FOLD server  was applhttps://saves.mbi.ucla.edu/, [https://prosa.services.came.sbg.ac.at/prosa.php, [http://molprobity.biochem.duke.edu/, [The accuracy of the protein structure model was evaluated using ERRAT , the Z-osa.php, ), and thke.edu/, ).http://haddock.science.uu.nl/services/HADDOCK2.2). Consensus prediction of interface residues in transient complexes (CPORT) (https://milou.science.uu.nl/services/CPORT/) was applied to accurate prediction of amino acid residues incorporated in the binding [For binding of class-I and -II epitopes to their HLA alleles, molecular docking studies were performed by HADDOCK 2.2 tool ( binding . Dockinghttps://web.expasy.org/protparam/) [https://pepcalc.com/) and the epitopes’ hydrophobicity was predicted by PEPTIDE 2.0 (https://www.peptide2.com/N_peptide_hydrophobicity_hydrophilicity.php). Epitope conservancy was checked via BLASTP and NCBI conserved domain search.The ProtParam tool (tparam/)  was usedhttp://webclu.bio.wzw.tum.de/cgi-bin/stride/stridecgi.py). The structures and location of the epitopes were depicted by the Web3DMol server (http://web3dmol.net/). The simplified molecular-input line-entry systemhttps://www.novoprolabs.com/tools/convert-peptide-to-smiles-string) and used to depict 2D structures of the peptides.The secondary structure prediction was done by the Stride Web interface  web servers.Antigenicity prediction of the final epitopes was carried out by the Vaxijen v2.0 , and ANT cell epitopes were predicted and several analyses were conducted to screen the predicted epitopes as follows: (1) excluding duplicate epitopes and choosing one epitope from an epitope cluster, (2) choosing high-scored epitopes and/or the epitopes predicted by more prediction tools, (3) weighing positivity in IFN-γ production and higher immunogenicity score for class-II and class-I epitopes, respectively, as preferred criteria of epitope selection, and (4) excluding allergens, toxic epitopes, and the epitopes with human similarity of 90% or more. By employing these analyses, the dominant class-I and class-II epitopes were chosen, and also their class II/I windows were found by comparing both classes with each other.The number of initially predicted class-I and class-II epitopes were 608, and 441, respectively, which were reduced to 92 and 62 following clustering and 22 and 27 after screening strategies  were modeled. The overall quality factor for Ep1, Ep2, and OMPK, as determined by ERRAT, was 79.21, 73.13, and 89.67, respectively. The Ramachandran plot showed that most amino acid residues in all three molecules  were located in the allowed and/or favored regions  and HLA molecules (receptors), ligand-receptor docking was performed by the HADDOCK tool. The docking scores of our class I epitope-HLA and class II epitope-HLA were higher than the docking scores of HLA combined with their original peptides (controls), showing the high affinity of our epitopes to their specific HLA molecules Table .E. coli, in vivo. The peptide is poorly water-soluble, but it is stable with a net charge of zero. The Ep2 (GKHISPDTRLYLGIE) peptide  had 33.33% hydrophobicity and a pI of 6.75. Its estimated half-life was > 30 h in mammalian reticulocytes in vitro and > 10 min in E. coli, in vivo. The peptide is good water-soluble, but it is unstable with a net charge of 0.1.Table Both Ep1 and Ep2 peptides were predicted to be antigens by the Vaxijen server, while neither of them was predicted to be an antigen by the ANTIGENpro server Table .Klebsiella pneumoniae.Both peptides are mainly composed of turns and they were both located on the lateral surfaces of the OMPK structure Fig. . The pepA. baumannii infections through both cell-mediated and humoral immunity. OMPK was able to trigger both humoral (IgG antibodies) and cytokines responses, which provided defense against a fatal challenge with A. baumannii [Previous research on OMPs has shown that these proteins can trigger defense against aumannii , 16, 38.aumannii , 16. MF5aumannii –41. In aaumannii –46.A. baumannii infections likely involves significant contributions from humoral immune responses, including IgM, IgG, and IgA [A. baumannii infections are still poorly understood. According to a small number of experimental studies, A. baumannii infection severity was linked to higher levels of Th1 inflammatory cytokine responses and lower levels of IL-10, suggesting a protective effect of Th2 immune responses [A. baumannii infections are still unknown [A. baumannii vaccine candidates. They were both immunogenic, non-toxic, non-allergen, and non-similar to the human proteome. In addition, they contain both class I and II T cell epitopes that were found to bind to their HLAs with high affinity. Ep1 was predicted to be able to produce IFN-γ while Ep2 could not, but it should not be assumed as a negative criterion since as mentioned above, the role of Th1 cytokines such as IFN-γ is not clearly understood in protection against A. baumannii [Host defense against  and IgA . The T cesponses , 49. How unknown , demandiaumannii .In the present study, the modeled 3D structures underwent verification using in silico tools. The majority of amino acid residues in our modeled protein (OMPK) and its final epitopic regions (Ep1 and Ep2) were located in the allowed/favored regions of the Ramachandran plot, indicating the high quality of our models. Furthermore, we utilized the ERRAT assessment to identify any potential anomalies in the structure , which sA. baumannii [The results of the present study showed that Ep1 and Ep2 peptides have different physicochemical properties so Ep1 is stable, poor water-soluble, more hydrophobic, and with a lower estimated half-life than Ep2. Being more hydrophilic is indicated by the negative GRAVY , as seenaumannii , 52, 53.A. baumannii-related diseases. The epitopes may be chemically synthesized or cloned/expressed and utilized as radiochemical probes in designing diagnostic kits [Both epitopes identified in this study are located on the surface of the OMPK protein, making them easily accessible to antibodies. This feature, in addition to their potential as vaccine candidates, also makes them suitable candidates for the diagnosis of tic kits .K. pneumoniae, so they may induce immune responses against K. pneumoniae as well.The conservancy study of the Ep1 and Ep2 peptides showed that they do not belong to a conserved domain superfamily, but they are highly conserved among Acinetobacter species, hence it may induce immune responses against many species of this genus. Also, both peptides had 100% similarity to a sequence of ion channel protein TSX of A. baumannii vaccines has increased significantly. OMPA [A. baumannii that have been studied in silico. Collectively, the results of our study, along with previous research provide promising prospects for the development of a suitable vaccine based on OMPs to combat A. baumannii infections in the future.In recent years, the number of bioinformatics studies on ly. OMPA , 56, Dcaly. OMPA , NucAb [ly. OMPA , and Tonly. OMPA  can be mA. baumannii OMPK high immunogenic class-I and class-II T cell epitopes, which can be utilized in future research. Additionally, two high immunogenic peptides containing both T cell epitope classes as well as the necessary physicochemical properties for the development of an A. baumannii vaccine were introduced. It is recommended to do an in vitro/in vivo investigation of these peptides to determine their true efficacy and efficiency.Altogether, we identified the Limited immunity due to several factors such as easy degradation and their recognition difficulty is linked to epitope-based vaccinations, such as our suggested Ep1 and Ep2 peptides. Conjugating them with conventional adjuvants and/or novel built-in adjuvants, such as new biomaterials or carriers, is one efficient technique to bypass this limitation . AnotherAdditional file 1: Table S1. The list of HLA alleles used for A. baumannii OMPK T cell epitope prediction.


Thermoplastic starch (TPS) has emerged as an essential alternative to produce environmentally friendly packaging; however, retrogradation is a disadvantage that affects its shelf life. This study analyzed the co-plasticizing effect of isosorbide on the mechanical, thermal, physicochemical, and microstructural properties and the retrogradation of films obtained by blown film extrusion from thermoplasticized starch with mixtures of glycerol and isosorbide in different ratios . The results showed that the higher concentration of isosorbide significantly increased the tensile strength; however, it reduced the elongation. Retrogradation modeled using the Avrami equation showed that the presence of isosorbide reduced the retrogradation rate (k) and modified the recrystallization mechanism (n). The relative crystallinity in the plasticized TPS films was reduced to 89%, and the adsorption significantly decreased. Isosorbide was very important in reducing the retrogradation of TPS. The best performance was obtained with the 2:1 ratio of glycerol/isosorbide due to the synergistic effect between the plasticizers. The results would allow tuning the properties of TPS films by combining glycerol/isosorbide in different ratios, which enables the design of materials tailored to potential application requirements. In recee chains . The usee chains , amines e chains , amides e chains , carboxye chains , ionic le chains , eutectie chains , amino ae chains , sugars e chains , and suge chains . Co-plase chains ,19.Glycerol is the most widely used plasticizer for obtaining thermoplastic starch due to its high boiling point, high solubility in starch chains, low cost, and availability . HoweverE), thermal stability, and resistance to retrogradation [E increased. At the same time, the glass transition temperature (gT) decreased, and the microstructure of the films was more homogeneous. By co-plasticizing sugar–glycerol, it is possible to reduce the cohesive forces of the polymer, demonstrating efficient plasticization [Several studies have investigated the mixing of plasticizers to transform thermoplastic starch ,11,20,21cization . In addicization .Isosorbide is a fused bicyclic diol and one of the three isomeric forms of 1.4–3.6 dianhydrohexitol characterized by two OH groups and two oxygen atoms intercalated on the carbon rings ,23. It iThe materials used were cassava starch with 12% water on a wet basis , commercial grade glycerol with 99.5% purity , and isosorbide with 98.5% purity .. The starch with the different proportions of plasticizers was mixed for 10 min in a KITCHEN AID  mixer and stored in hermetically sealed PET containers for 48 h [The starch was dried at 80 °C for 16 h until it reached a humidity of less than 2%. The dried starch was mixed with plasticizers: glycerol and isosorbide in a ratio of 70:30 (starch/plasticizer). For co-plasticization (glycerol–isosorbide), different mixtures were made in the proportions presented in Table 1for 48 h ,28,29. TThe pellets were extruded to obtain films by extrusion blowing with a single screw extruder coupled to a blow die. The temperature profile was 130, 157, 153, and 149 °C from the feeding zone to the die. The speed was 35 rpm. The films obtained were stored in a chamber at an average ambient temperature of 20 °C and relative humidity (RH) of 60%.The tests were performed according to norm ASTM D-882-10 on a universal machine  at a deformation speed of 25 mm/min. The jaw spacing was 50 mm. The product obtained was cut in the machine direction (DM) and transverse to the machine (DT), with dimensions 20 mm wide and 70 mm long. The tests were carried out at room temperature.3)2) and potassium chloride (KCl), respectively. All solutions were prepared with distilled water. Samples were weighed periodically every 24 h for the first 8 days and then at 15 and 30 days of storage. The percentage of moisture absorption (H) expressed in percent was calculated according to Equation (1) below, where tW is the weight at time t, and iW is the weight in weight of dry material:This method dried TPS samples cut to 20 mm × 50 mm in a forced convection oven at 80 °C for 24 h. The initial weight of each sample was taken, and the samples were immediately placed in a chamber with a relative humidity of 60% and 90%. Moisture contents of 60 and 90% were fixed using saturated solutions of magnesium nitrate  coupled to an ATR device. The spectra of all samples were recorded with 4 cmRC) was estimated using Equation (2) below, where AC corresponds to the crystalline area, and AA is an amorphous area:The crystallinity of the starch and TPS films was analyzed by X-ray diffraction. An XRD diffractometer  was used at a current of 30 mA and a voltage of 40 kV. Scans were performed at 2ϴ between 7 and 40°. Relative crystallinity (H). The degree of retrogradation (DR) was calculated by Equation (3) [tH is the melting enthalpy (J/g) of the sample after retrograding for different times, ∆H0 is the melting enthalpy (J/g) of the sample without retrogradation, and ∆∞H is the melting enthalpy (J/g) of the sample after retrograding for 30 days. The analysis of the retrogradation kinetics was carried out by applying the theory of Avrami, expressed in Equations (4) and (5) [DR is the degree of retrogradation, and k and n indicate the crystallization rate constant and Avrami exponent, respectively. The ln k corresponds to the intersection, and the n value is the slope of plotting Phase transitions were performed according to norm D3418-08 using a TA Instruments Q20 calorimeter . A scan was performed from −60 °C to 250 °C at a heating rate of 20 °C/min. The parameters of retrogradation kinetics were calculated from the enthalpy results (∆tion (3) :(3)DR=ΔHThe TGA was performed by norm ASTM E1131 (2008) using a thermogravimetric analyzer TA Instruments TGA Q50 . Samples of 4 to 6 mg were deposited in a platinum capsule inside the sample holder of the equipment. Heating was performed from room temperature to 600 °C at a heating rate of 20 °C/min in a nitrogen atmosphere.The morphology of the films obtained was analyzed on the surface using a Flield-emision scanning electron microscope (FE-SEM) Hitachi UHR 8010 . The samples were vacuum dried at a pressure of 25 psi and gold–palladium coated using a Quorum machine Q150-ES .p-value < 0.05) was used to evaluate the difference between the mean values of the mechanical properties of the films.The experimental results of the mechanical tension properties were analyzed by ANOVA. Tukey’s test . The E value was twice higher in the case of TPSi-10 and up to four times higher for TPS-i20 films. The same behavior was observed for the tensile strength (σ) values.The obtained results are compiled in Ɛ) decreased by 6% for TPS-i10 and 37% for TPS-i20; this indicates that the behavior of films with isosorbide tends to be stiffer, which is in agreement with the results reported by other authors [E, σ, and Ɛ of 55 MPa, 2.7 MPa, and 72.4%, respectively, for isosorbide-plasticized films obtained by extrusion and compression molding. The above values were higher than those observed for films plasticized with glycerol [E, σ, and Ɛ of 8.7 MPa, 2.7 MPa, and 142.4%, respectively, were reported. However, in the latter case, the TPS preparation technique was by casting. In both cases, the authors attributed this behavior to the compatibility of the bonds formed between plasticizer and starch [In contrast, the strain value   after 30 days of storage, while the strain (Ɛ) was reduced by approximately 80%; this suggests that the TPS-G materials underwent a higher degree of retrogradation, due to the reorganization of the structures that had been destroyed during starch extrusion and plasticization [The results show that the stiffness, ngth (σ)  of filmscization . In addicization . The strcization . As prevcization ,35.E value did not undergo considerable changes. In TPS-i20, the E value for day 30 was reduced by 70%. Tensile strength was the parameter most affected by time, with a decrease of 76 and 77% . With the 1:2 glycerol/isosorbide ratio, the elongation of the films increased with time and reached the highest value (203.3%). In general, the TPS-i10 films (glycerol/isosorbide ratio of 2:1) were the least susceptible to time-induced changes, which could indicate a lower degree of retrogradation. Among the polyols, sorbitol has been shown to slow the rate of recrystallization best in the TPS [H crystals, which contain more water molecules [In the TPS-i10 films, after 30 days of storage, the  the TPS  because olecules . Isosorbolecules ,31. Thisolecules .Few studies have reported the water-absorption capacity of starch-based materials plasticized with isosorbide. It has been reported that films plasticized with isosorbide showed lower water absorption than when glycerol was used . The lowOne of the functions of plasticizers is to break the hydrogen-bonding forces existing in native starch to convert it into a thermoplastic material ,40. Infr− 1, characteristic of starch [−1 corresponds to the stretching of the free OH groups of the starch structure [–1) when the starch was plasticized with glycerol. Other authors have observed this behavior in potato starch materials plasticized with glycerol [−1 in starch decreased in intensity in the TPS as the isosorbide content increased; this is related to less water absorbed by the amorphous regions of the plasticized starch [Three absorption bands were observed at 3312, 1149, and 997 cmf starch . The abstructure . It is atructure . This baglycerol . The addglycerol ; howeverglycerol ,17. The d starch  since thd starch , as veri−1 (−1 and two undefined shoulders at 1047 and 1016 cm−1), which could indicate the molecular interaction between plasticizer and starch. The bands at 1149 cm−1 and 1078 cm−1 correspond to the C–O stretching of the C–O–H group. The absorption band between 920 and 936 cm1 was assigned to the C–O bond stretching vibration of the C–O–C α bond between the anhydroglucose rings of native starch [−1 corresponding to the C–O stretching of the C–O–C group of the starch anhydroglucose ring was intensified [−1 is associated with plasticization [–1 was observed in the TPS, corresponding to glycerol functional groups [−1 decreased almost entirely when starch was plasticized, regardless of the type of plasticizer. The 1047 cm−1 absorption band is sensitive to the number of starch crystals [In the region from 1200 to 800 cm−1 b, changee starch . In the ensified ; this isensified , indicatensified . The abscization , and itscization . A new b−1 is not directly related to plasticization [−1, indicating an increase in recrystallization and an increase in the intermolecular strength of hydrogen bonds between starch chains [−1 shifted towards a slightly higher wavenumber for all samples, whereas the absorption bands at 1078 and 1016 cm−1 did not change substantially with time. Although it has been reported that, during TPS aging, the height of these three bands increases [The FTIR spectra of the TPS were evaluated on day 30, and their typical absorption bands did not change drastically. Although the interaction associated with the wavelength range between 3800 and 2800 cmcization , in sampncreases ,48,49, nAA) was higher. In contrast, the crystalline area (AC) decreased , a small peak at 24° was observed, attributed to residual crystallinity due to incomplete destructuring of native starch granules, which is also evident in the analysis of SEM pictures. Contrary to TPS-G films, TPS-i materials presented a single intense peak located around 18°, which is associated with Eallinity , a produructures . Accordiructures . The sizructures .H) did not change with storage time. Crystallinity  and the heat or melting enthalpy (∆mH), due to the interactions created by the addition of isosorbide, through hydrogen bridges between the starch and plasticizer chain [The partial substitution of glycerol by isosorbide drastically modified the melting temperature  were close to 1, indicating a high fit of the experimental data in the model. However, the fit for the TPS-G treatment was lower, possibly due to the high moisture absorption capacity mainly produced in the first days of storage, which causes a rapid retrogradation [n) provides qualitative information about nucleation and crystallite growth [n were less than 1 in all samples, being lower for TPS-G. In TPS-i10 and TPS-i20 films, the exponent n was almost three times higher than TPS-G, which shows that the incorporation of isosorbide influenced the nucleation mechanism and crystallization processes during TPS retrogradation.The TPS samples’ retrogradation behavior d was evae growth . The valn less than one could indicate that the formation of nucleation points followed an instantaneous mechanism during starch recrystallization, and the latter occurred mainly in the initial stage of storage [k) decreased drastically with the addition of isosorbide, with similar values for TPS-i10 and TPS-i20 films, indicating a reduction in the retrogradation rate [n and k values in order of magnitude have been reported for glycerol-plasticized and SiO2-added TPS films obtained by compression molding [k values calculated based on the storage modulus were much lower. However, the k for the TPS containing glycerol was higher than the other plasticizers [These observations are consistent with the XRD results, suggesting the formation of different crystalline structures according to the plasticizer. Values of  storage . The cryion rate ; this ision rate , as evidion rate ,67. Simi molding . In filmticizers , which cThermogravimetric analysis was performed to study the influence of the plasticizer on the thermal degradation of TPS films co-plasticized with glycerol/isosorbide. The results were plotted using TGA curves . The the10%, T50%, and T90%, indicating the temperature at which 10%, 50%, and 90% mass were lost, respectively. Tmax is the maximum degradation temperature. The residue (%) is the fraction remaining after heating to 591.5 °C (maximum test temperature). The most significant changes were observed in the first stage of thermal degradation (T10%, integrated plot in 10% for films containing isosorbide could be related to the lower volatility of this plasticizer but also suggests higher thermal stability of TPS-i10 and TPS-i20 films [The data extracted from the TGA curves are summarized in 20 films .Previous studies indicate that isosorbide effectively integrates starch and plasticizer into a single phase, where the plasticizer molecules are trapped inside the starch ; this is10% decreased with time, indicative of a loss of thermal stability of the materials, the T10% of the films containing isosorbide remained above the T10% of the films containing only glycerol as the plasticizer; this suggests that the initial starch–isosorbide interactions were more stable than the starch–glycerol interactions. Lower water absorption of TPS-i films favors this behavior, since a low amount of water reduces the ability of starch chains to reorganize [The thermal stability of the films as a function of time b–d showsorganize ,61.The surface morphology of the glycerol/isosorbide co-plasticized TPS films was studied by scanning electron microscopy . A compaThe plasticizer’s processing and presence destructured most of the starch granules. However, residual starch granule structures were observed outside the matrix b,d,f andThis study obtained environmentally friendly bio-composites using raw materials of renewable origin, such as cassava starch. In addition, implementing a co-plasticization system with glycerol and isosorbide allowed complementing of the plasticizing properties of glycerol and the anti-retrogradation effect of isosorbide to obtain thermoplastic starch-based materials. Isosorbide promoted changes in the mechanical tension properties of TPS as a function of the glycerol/isosorbide ratio. Although the elongation decreased with an increasing amount of isosorbide, the presence of glycerol in the co-plasticization system allowed for obtaining a sufficiently flexible material with higher re-strength.H-type crystalline structures, which are more stable over time and reduce the effects of retrogradation. A glycerol/isosorbide ratio of 2:1 is effective in achieving adequate starch plasticization, comparable to other plasticizing systems. This evidence suggests a synergistic effect between glycerol and isosorbide.A partial modification of the recrystallization mechanism was identified with isosorbide, promoting the formation of predominantly EThe results obtained are promising and helpful in the search for biodegradable materials for different applications; however, other parameters should be evaluated according to the specific application, such as oxygen or water vapor permeability, color, and transparency in the case of food packaging.


Suggested citation for this article: Breedlove B. Notice to readers about the cover. Emerg Infect Dis. 2023 Dec [date cited]. https://doi.org/10.3201/eid2912.AC2912Alice’s Adventures in Wonderland, written by Lewis Carroll  and illustrated by the British artist Arthur Rackham.Appearing on this month’s cover is an image titled “The Pool of Tears.” The image comes from the 1907 edition of Emerging Infectious Diseases is instead using this space to make a call for About the Cover submissions from readers interested in the intersection of art and science, medical history, and biographical sketches. Those considering this invitation should first read the information on our website at About Cover Art (https://wwwnc.cdc.gov/eid/page/about-cover-art).Rather than following our usual practice of publishing a short essay to accompany the cover image, this month Cover essays are intended to relate science and the human condition, or how people perceive and cope with infection and illness. EID seeks for its cover images and their accompanying essays to evoke compassion for human suffering and to expand the science reader's literary or artistic scope, provide context related to the theme of each month’s issue, and link the art with public health and science. Although we want scientific information mentioned in about the cover essays to be accurate, these are not research articles. Each issue’s theme is typically decided and assigned two to ten months before the publication date. Typical themes include zoonotic infections, vectorborne infections, respiratory infections, emerging viruses or emerging pathogens, fungal infections, bacterial infections, parasitic diseases, or some variation of those general themes. EID also publishes issues with themes related to specific diseases or health topics.All genres of art from all cultures, historical periods, and geographic areas are considered. Potential contributors may wish to contact the journal before submitting an essay to see if their proposed image and idea for an essay are within the scope and interest of the journal. Contributors must adhere to a number of guidelines and tips for About the Cover submissions:Essays are a maximum of 900 words text.No abstract or summary line is needed.First author biography is not required.One high-resolution image is used on the cover of the journal; up to two secondary images might be permitted to accompany the essay.Authors are responsible for obtaining any approvals or releases for use of images that are not in the public domain, copyrighted, or otherwise restricted.https://wwwnc.cdc.gov/eid/past-covers). Pay attention to format and tone of the essays.Examples of cover essays may be found in every issue of the journal from 2002 forward  along with each essay.Submit the EID Author Checklist (All essays are peer reviewed, and the editor-in-chief makes the final decision regarding publication.eideditor@cdc.gov. Meanwhile, we hope that many of you are thinking about what you might have written about this month’s cover image, “The Pool of Tears,” and how it could relate to zoonotic infections.If you would like to consult about any aspect of an About the Cover essay for upcoming issues of EID, contact the journal at


Philip Alexander, MD, is a native Texan, retired physician, and accomplished musician and artist. After 41 years as an internal medicine physician, Dr. Phil retired from his practice in College Station in 2016. A lifelong musician and former music professor, he often performs as an oboe soloist for the Brazos Valley Symphony Orchestra. He began exploring visual art in 1980, evolving from pencil sketches—including an official White House portrait of President Ronald Reagan—to the computer-generated drawings featured in this journal. His images, which first appeared in this journal in the spring of 2012, are his own original creations.Methodist DeBakey Cardiovascular Journal, submit your creation online at journal.houstonmethodist.org as a “Humanities” entry.If you would like to see your art published in the


International Wound Journal. 2023; 1–9, https://doi.org/10.1111/iwj.14101. The above article from International Wound Journal, published online on January 30, 2023 in Wiley Online Library (wileyonlinelibrary.com) has been retracted by agreement between the journal Editor in Chief, Professor Keith Harding and John Wiley & Sons, Ltd. The retraction has been agreed due to unattributed overlap between this article and the following article: Manzano‐Nunes, A. Gomez, D. Espitia et al., A meta‐analysis of the diagnostic accuracy of chest ultrasound for the diagnosis of occult penetrating cardiac injuries in hemodynamically stable patients with penetrating thoracic trauma. Journal of Trauma and Acute Care Surgery, 2021, 90(2):p 388–395, https://doi.org/10.1097/TA.0000000000003006.Yan, T, Xie, W, Xu, M. Effect of chest ultrasound compared with pericardial window for the diagnosis of occult penetrating cardiac wounds in hemodynamically stable subjects with penetrating thoracic trauma: A meta‐analysis.  International Wound Journal. 2023; 1–9, https://doi.org/10.1111/iwj.14101. The above article from International Wound Journal, published online on January 30, 2023 in Wiley Online Library (wileyonlinelibrary.com) has been retracted by agreement between the journal Editor in Chief, Professor Keith Harding and John Wiley & Sons, Ltd. The retraction has been agreed due to unattributed overlap between this article and the following article: Manzano‐Nunes, A. Gomez, D. Espitia et al., A meta‐analysis of the diagnostic accuracy of chest ultrasound for the diagnosis of occult penetrating cardiac injuries in hemodynamically stable patients with penetrating thoracic trauma. Journal of Trauma and Acute Care Surgery, 2021, 90(2):p 388–395, https://doi.org/10.1097/TA.0000000000003006.Yan, T, Xie, W, Xu, M. Effect of chest ultrasound compared with pericardial window for the diagnosis of occult penetrating cardiac wounds in hemodynamically stable subjects with penetrating thoracic trauma: A meta‐analysis.


Nanocomposite film was formed from UTPS/ISC2 in presence of stabilized iodinated cellulose nanocrystals UTPS/ISC2/SICNCs via gelatinization at a temperature of 80ºC. Water solubility and water vapor release were studied amongst the water barrier features. The fabricated starch-based composite films were evaluated utilizing Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electronic Microscope analysis (SEM), surface area, and tensile measurements. The adsorption of crystal violet (CV) dye onto produced samples was examined in an aqueous solution. The findings revealed that the UTPS/ISC2/ISCNCs has 83% crystal violet elimination effectiveness. Moreover, the adsorption isotherms were assessed and figured out to vary in the order of Langmuir > Temkin > Freundlich > Dubinin-Radushkevich.Modification of starch is a potential basic research aiming to improve its water barrier properties. The general purpose of this study is to manufacture cross-linked iodinated starch citrate (ISC) with a degree of substitution (DS) ≈ 0.1 by modifying native corn starch with citric acid in the presence of iodine as an oxidizing agent. Thermoplastic starch (TPS) was generated with urea as a plasticizer and blended with various concentrations of ISC of  (wt/wt) to obtain (UTPS/ISC In recent years, biodegradable plastic has emerged as one of the most inventive and rapidly growing segments of the plastic markets . The mosResearchers have found that iodine is an ideal acylating agent in the acetylation of polysaccharides such as dextran, cellulose, and starch . Iodine Industrial wastes with various pollutants have a significant negative impact on biodiversity, the ecological environment, and the characteristic ocean activities. In the midst of these impurities, synthetic dyes such as crystal violet (CV) dye are widely used for cotton, dyeing the silk, manufacture of paints, and printing inks. Crystal violet is carcinogenic, non-biodegradable, and can persist in various environments . FlotatiThe purpose of the current study is to investigate the physicochemical characteristics of composite films that have been synthesized based on starch as a biopolymer, urea as a plasticizer, citric acid, iodine, and CNCs for starch modification treatment to ameliorate starch stability in aqueous solutions as a substitute for the treatment of crystal violet dye from wastewater. Batch adsorption experiments were carried out to evaluate the efficacy of the composite films created at various initial dye concentrations.Corncob feed stocks were obtained from nearby areas in the maritime sub-tropical district (Nile Delta). Cobs were threshed to remove the straw and chaff, then dried to less than 10% moisture content and sieved to eliminate any foreign residues. In an air machine, the crushed material was sanitized to be used as a source for CNCs manufacturing. Citric acid (97%), urea (99%), ethanol (95%), sodium thiosulfate (99%), and iodine crystals (98%) were obtained from Sigma-Aldrich Company. Crystal violet dye was purchased by the Merck Company. Corn starch (S) is a type of starch derived from corn that can be available commercially.wt/wt) were heated at 100 °C for 15 min in a muffle furnace, cooled, and mixed with 2 mL of saturated sodium thiosulfate solution with stirring till the color of the mixture had been changed to colorless, indicating the alteration of iodine to iodide. The mixture was gushed into 50 mL of ethanol and stirred for 1 h. The produced iodinated starch citrate (ISC) was filtered, rinsed with distilled water and ethanol to uptake non-reactive citric acid, and dried overnight in a vacuum oven at 60 °C.The solid–solid interaction technique using iodine as an oxidizing agent was mentioned in the starch modification procedure as described by Biswas et al.  with a mB is the volume of HCl (mL) accustomed to titrate the blank sample, VS is the volume of HCl (mL) applied to titrate the ISC sample, M is the concentration of the HCl (mol.L−1), 43 is the molar mass of the acetyl group (g.mol−1), and m is the mass (g) of the ISC . Every half hour, each sample was weighed for 3 h. Using the following equation the mean value of the water vapor absorption was calculated from three intervals:t is the weight of the sample after "t" time and Wº is the initial weight of the sample film. The relative humidity resulted from the absorption of saturated moisture produced by water vapor at 100 °C was ≈ 95%.The absorption of water vapor was measured based on the moisture absorption technique. Moisture absorption examination was performed according to the application of the following approach, each film was cut into 2 × 2 cm squares, then weighed and hung from a wire. The wire was suspended in a beaker containing evaporated water at 100 °C. Water vapor release was achieved by varying the weight of each sample [S, UTPS, UTPS/ISCThe water solubility of each film was determined using the dry matter percentage of the soluble film after immersion in distilled water. The fabricated samples were cut and dried at 100 °C to determine the dry weight of the material. Each sample was sealed in a 40 mL beaker of distilled water and incubated for 2 h at 25 °C with periodic delicate shaking. The samples were removed from water by filtration and insoluble dry matter was determined by drying in an oven at 100 °C up to a constant mass . The per−1 along with four resolutions averaged over 40 scans.The influence of plasticization and modulation on the functionality of the surface of starch-based films was analyzed through FT-IR spectroscopy employing the KBr pellet approach on an FT-IR NICOLET 8700 spectrometer  in the spectral range of 400–4000 cmThe surface morphologies of the fabricated composite film samples were checked by applying QUANTA FEG 250 ESEM (Japan).2 at 77K with a surface area analyzer model .The specific surface area (Brunauer–Emmett–Teller (BET) method) and the features of the pores of the synthesized composite films were evaluated by adsorption–desorption of N2, and UTPS/ISC2/SICNCs composite films. Consequently, stock solutions with diverse concentrations were prepared by dissolving (10–100) mg of CV dye in 1 L of distilled water. 100 mg of fabricated samples were administered at pH (6.5) using 0.1 M HCl and 0.1 M NaOH with 10 mL of CV dye solution. To attain equilibrium, every sample was kept in a rotating shaker  at 220 r.p.m, for 24 h at room temperature. Preliminary tests showed that 24 h was adequate to reach equilibrium. The UV–visible absorption spectra of the supernatant solution were analyzed employing a UV–visible spectrophotometer (Type UV-2401PC) in a 1 cm quartz cuvette to check the characteristic absorption peaks of CV dye at a wavelength of 590 nm. The equilibrium adsorption amount, qe (mg/g), was determined according to the following equation:o and Ce (mg/L) are the initial and equilibrium concentrations in the liquid phase, V (L) is the volume of the equilibrium solution, and m (g) is the mass of the adsorbent. The percentage of dye removal from the aqueous solution was conditioned by applying the following equation, where R is the removal efficiency of the dye:The aim of this work is to study the possibility of treating aqueous solutions contaminated with crystal violet dye (CV) by UTPS, UTPS/ISCe is the equilibrium dye concentration (mg/L), qe is the adsorption capacity at equilibrium (mg/g), qm is the maximum adsorption capacity related to complete monolayer coverage on the surface (mg/g); and KL is the Langmuir constant (L/mg), indicating the nature of adsorption. The main feature of the Langmuir isotherm can be stated in terms of the equilibrium parameter RL, which is the dimensionless constant known as the separation factor [o is the initial concentration for maximum removal efficiency.The Langmuir isotherm is used to valuate the coverage of a monolayer with uniform adsorption energies onto the surface without adsorbate transmigration into the surface plane , 26. It n factor . Co is tL value indicates whether the adsorption feature is favorable if 0 < RL ≤ 1, unfavorable if RL > 1, linear if RL = 1, and irreversible if RL = 0 [The Rf RL = 0 , 28.F (mg/g)(L/mg)1/n represents the Freundlich isotherm constant, and n represents the adsorption intensity constant. At n = 1, the separation between the two phases is independent on the concentration. If the value of 1/n is < 1, this indicates that the adsorption is normal. In contrast, 1/n > 1 demonstrates cooperative adsorption [The Freundlich isotherm estimates the adsorption properties of the heterogeneous surface , 30. It sorption .T is the equilibrium constant of the Temkin isotherm (L/mg) related to the maximum binding energy, bT is the Temkin isotherm constant corresponding to the heat of adsorption (J/mol), R is the constant of universal gas (8.314J/mol.K), and T is the temperature (K).The Temkin isotherm equation presumes that the heat of removal of full molecules in the layer could decrease linearly instead of logarithmically with coverage due to adsorbent-adsorbate interactions. This model is given as:mDR (mg/g) is the adsorption capacity of saturation theory (mg/g), KDR (mol2/J2) is the Dubinin-Radushkevich isotherm constant, and ε is the potential Polaniyi (J/mol). This model is used to differentiate physical and chemical adsorption through its average free energy, E (J/mol), which is required to remove an adsorbate molecule from its position at the adsorption site to infinity. It is calculated using the equation:Dubinin-Radushkevich assesses both homogeneous and heterogeneous surfaces with a Gaussian energy distribution. It features a compelling specific parameter for the average free energy, which is utilized to differentiate physical and chemical adsorption. The equation is given as follows:2, and UTPS/ISC2/SICNCs composite films. The tensile test of the samples was carried out in a humid environment for 5 days. The fiber mats (30 mm length × 10 mm width × 2 mm breadth) were adhered to a paper fixing tab with an epoxy adhesive with a gauge length of 20 mm and a tensile speed of 2.5 mm/min. When the sample was firmly gripped and cut from both sides of the tab, the tensile test began. The mean values of tensile strength, modulus of elasticity, and elongation at break were estimated at least 3 times for each sample [2 or MPa), F is the load (N), and A is the cross-sectional area (mm2).◦ is the actual sample length (mm), and L is the sample length at breaking point (mm). The modulus of elasticity, E, is a measure of the ability of a material to tolerate longitudinal changes under tension or compression. It can be represented by a linear regression analysis of the initial linear section of the stress–strain curves, applying the formula illustrated as follows:E is Young’s modulus (MPa), and tensile strength can be determined from the maximum stress point .Uniaxial tensile examination with a comprehensive  machine has been upgraded to UTPS, UTPS/ISCh sample . The ten2, UTPS/ISC4 and UTPS/ISC6 ranged from 14 to 36.5%, from 15.4 to 55.2% and from 13.5 to 53% respectively, UTPS/ISC2 had the lowest amount of the water vapor absorption. From Fig. 2 implying a more controlled absorption property as a result of plasticization and the modification of ISC. This phenomenon was explained by the presence of urea that enfolds the internal polymeric chains of the starch increasing the intermolecular forces between the polymer chains and decreasing the free volume, hence it can prevent water molecules from spreading through the films. Furthermore, the hydrophobic ester groups in the cross-linked starch citrate introduced new intra-molecular bonds that limited the amount of water vapor that can be absorbed in starch [2 raised the water vapor (%) to 71.4 after 2 h revealing their poor release profile. This was assumed to be related to the high hydrophilicity and porosity of SICNCs, which favored the absorption of water molecules and contributed to an increase in the water vapor permeability of the film. Most non-absorbency applications are impeded by poor barrier properties of this composite film, however low water vapor absorption films have better barrier properties and good packaging applications [By assessing the amount of water vapor that can pass through the pores and boundary surface of the bio-polymer film, the experimental study on the water vapor absorption aids to understand the barrier behavior of as-synthesized films . Table 1n starch , so it cications .Table 1W2. This result revealed that the modification of starch with urea and cross-linking (ISC2) strengthened the bonds between adjacent starch chains, thereby reducing their tendency to split during solubility [The water solubility of films is one of the most important properties for understanding biodegradable packaging. Starch applications are generally limited because they are unstable with changes in temperature, water, pH, and shear forces . Water ilubility . Ester glubility . Furtherlubility .Fig. 2Wa2/SICNCs revealed insoluble properties and no water absorption at room temperature, owing to their rigidity against water solubility as measured and the creation of a three-dimensional network of cellulose via the generated hydrogen bond between starch and SICNCs. This network limited the solubility of the polymer, caused strengthening of the network, and reduced the contact between water and the produced film. The results highlighted the composite films resistance to humidity, temperature, and water solubility.Surprisingly, UTPS/ISC2 are revealed in Fig. −1. The C-O stretching vibration band has been assigned to these peaks. A peak at 1650 cm−1 was attributed to tightly bound water by a strong H-bond, and the starch biopolymer’s characteristic absorption bands were in the range of 2800–3000 cm−1 due to the -CH stretching of starch moieties. Another exceedingly broad band at 3292 cm−1 was associated with the vibration of the linked hydroxyl group (O–H) [−1. ISC2 showed similar FT-IR spectra with increasing intensity of the characteristic peaks at 1650 cm−1 and 1020–1150 cm−1 owing to carbonyl group generation (C = O), and C-O stretching vibration, respectively [−1 of ISC2 was relevant to the stretching of the –CH3 of acetyl groups. The expansion in the band intensity of ISC2 has been attributed to new citrate functional groups or to new intramolecular hydrogen bonds among the hydroxyl groups concerning the starch and the carbonyl group regarding citrate.The introduction of extra functional groups into the starch-based films was validated by FT-IR spectroscopy. The spectra of native starch (S) and ISCup (O–H) , whereasectively . The ban2 and UTPS/ISC2/SICNCs are displayed in Fig. −1 in the UTPS spectra, represented by the OH vibration. Urea has a distinct absorption band in the regions at 3432 cm−1 which is characteristic of N–H amide stretching, and N–H amide bending with characteristic peaks found at 1595 cm−1 and 1621 cm−1, respectively [−1 showed the strong stretching of the -C-N groups, this peak emerged sharper with the incorporation of ISC2 in the UTPS/ISC2 and UTPS/ISC2/SICNCs spectra, which explained the association of ISC2 in the starch plasticizing system [−1 appeared sharper after incorporation of ISC2 supporting its contribution to the starch chains and urea. The spectra of UTPS/ISC2 and UTPS/ISC2/SICNCs depict the typical bands of urea and starch citrate, for a peak around 2800 cm−1 that indicates the C-H stretching of an aliphatic methyl group with a noticeable reduction in its intensity, suggesting new elongation of the backbone chain after mutation with ISC2 and SICNCs. For UTPS/ISC2/SICNCs, the absorption peak at 3335 cm−1 became slightly sharper for the hydroxyl group due to the hydrogen bond interaction when starch and SICNCs were included together in the nanocomposite manufacture. With the absence of the functional groups of lignin and hemicellulose, the characteristic band of the cellulose pyranose ring was evident at 1070 cm−1, ensuring the efficiency of the delignification and bleaching pretreatment steps [The FT-IR spectra for UTPS, UTPS/ISCnt steps , 45. Thent steps .Fig. 4FT2 particles still persist intact with the same particle size and shape, but with wrinkled cores and lengthy tortuous grooves that may be attributable to the exposure to iodine and citric acid. Iodine, acting as an oxidizing agent, caused defects on the surface of the starch, making it rougher, while citric acid induced eco-corrosion on the starch surface. The minor change in the micrographs was assumed to the increase of D.S which may damage the intermolecular hydrogen bonds and reduce the resilience of starch, as interpreted by Sha et al. [Figure a et al. . As demo2 in Fig. 2 that caused chain fragmentation of starch molecules. Thus, it decreased the interaction between the molecular chains of the starch improving the plasticization process of starch [2 film observed in Fig. The micrographs of starch-based composite films are present in Fig. f starch . NeverthBET, m2/g), total pore volume , and mean pore diameter  were evaluated for the starch-based films according to the Brunauer–Emmett–Teller (BET) equation, and the data are given in Table Specific surface area evaluation SBET, m/g, total2 and UTPS/ISC2/SICNCs were ,  and , respectively representing the inhibitory effect of ISC2 and SICNCs on UTPS assembly. With respect to the average pore diameter, the results revealed that the produced starch-based films were consistent with a microporous nature. The reduction of microporosity and SBET with the exfoliation of ISC2 and SICNCs in the polymer matrix confirmed their effective involvement in enhancing the plasticization of starch-based films, as addressed in SEM micrographs.Interestingly, the surface area and total pore volume of UTPS, UTPS/ISC2/SICNCs, as obvious in Table 2 (66.2%) was obtained at 40 mg/L. This phenomenon can be clarified by the certainty throughout the initial phases of adsorption, a significant number of free sites were available on the adsorbent surface, but these sites were eventually occupied by dye molecules and the adsorption capacity remained constant after equilibrium [2/SICNCs film was more effective in remediating moderately concentrated CV from wastewater than other produced starch-based composite films. The adsorption of the CV dye on the manufactured starch-based composite films proceeded via electrostatic interactions with the highly negatively charged binding sites(FT-IR section), which was greatly enhanced after the incorporation of ISC2 and SICNCs into the UTPS film. Therefore, the adsorption of the CV dye was related to the surface chemistry rather than to the textural properties. The results of the percentage removal of CV by fabricated starch-based composite films are presented in Fig. As shown in Fig. ilibrium . As estailibrium , adsorpt2 were displayed individually. Langmuir is more applicable than Freundlich, Temkin and Dubinin-Radushkevich models in terms of R2 values. The Langmuir model anticipated the establishment of a regular and homogeneous monolayer adsorbate on the outside surface of the adsorbent. Additionally, the RL values were lower than 1, indicating that the dye was being adsorbed favorably. The values of n from the Freundlich parameters were greater than unity, indicating that the process of CV adsorption onto the fabricated samples was effective. The positive Temkin constant (bT) values  of UTPS, UTPS/ISC2, and UTPS/ISC2/SICNCs, respectively for the Temkin isotherm revealed that the adsorption was exothermic [2, and UTPS/ISC2/SICNCs are 0.353, 0.408, and 0.707 kJ/mol, respectively supporting the suggested dominating mechanism of physisorption [2 values seemed to follow the order; Langmuir > Temkin > Freundlich > Dubinin-Radushkevich.Some isotherm models have been utilized to evaluate the isotherm performance for CV adsorption and the fitting of the experimental data. Langmuir, Freundlich, Temkin, and Dubinin-Radushkevich were among them. In Figs. othermic . The valsorption . Table 4The dispersion and compatibility of one component with another are critical, as these factors have a substantial impact on evaluating the mechanical features of composite materials. A significant advancement in the mechanical characteristics can be achieved by establishing a homogeneous dispersion of the reinforcing filler in the matrix associated with good interfacial adhesion , 61. The2 and SICNCs merged into starch films is shown in Fig. 2, and UTPS/ISC2/SICNCs. This modification caused an increase in the hardness of the starch-based films produced. The tensile strength decreased by 35% with UTPS/ISC2, but it slightly increased with UTPS/ISC2/SICNCs, with a ratio of 4.2% comparable to UTPS, and the most rigid structure was obtained with UTPS/ISC2/SICNCs. Since the starch acetylation reaction advocates incomplete hydrolysis, it also breaks the glycosidic bond on the starch molecular chains. As a result, the molecular weight of starch reduced resulting in a shorter molecular chain, thus the UTPS/ISC2 tensile strength decreased. On the contrary, the elongation at break increased dramatically from 30.3 to 42% for UTPS and UTPS/ISC2, adequately and remained constant for UTPS/ISC2/SICNCs, as seen from Table It is known that a high value of Young’s modulus (E) indicates low elasticity and high hardness properties . Accordi2/SICNCs acted as a spacer and prevented the starch chains from getting too close, resulting in more interstitial space between the starch molecules [2 film, the SICNCs can capture this space and enhance the interaction with polymers, increasing the integrity of the film structure, as a consequence its tensile strength raised. Moreover, the enhancing effect of SICNCs on UTPS could be related to good dispersion and strong compatibility between UTPS and SICNCs in the film matrix due to the presence of hydroxyl groups and the formation of hydrogen bonds. Additionally, the nanosized cellulose allowed good surface contact with the starch matrix. Nevertheless, the chemical similarities between starch and cellulose can lead to a significant intermolecular hydrogen bonding interaction between the molecules [2/SICNCs compared to other starch-based films.The acetyl group in UTPS/ISColecules . Compareolecules . In respolecules , which r2 and UTPS/ISC2/SICNCs. The inclusion of ISC2 and SICNCs into TPS films improved the water stability of biopolymer starch. The higher water vapor absorption rate in UTPS and UTPS/ISC2, the greater the controlled barrier property. UTPS/ISC2/SICNCs film demonstrated insoluble characteristics and no water solubitity at room temperature in spite of its poor water vapor absorption profile. Furthermore, the reduction in SBET measurements associated with the exfoliation of ISC2 and SICNCs within the polymer matrix illustrated their ability to improve the plasticization of starch-based films, as evidenced by SEM micrographs revealing an extensive destruction of the morphology of the produced starch granules. The addition of ISC2 and SICNCs fillers to UTPS enhanced the Young’s modulus from 29.3 to 44.2 and 79 MPa for UTPS/ISC2 and UTPS/ISC2/SICNCs respectively, resulting in a higher hardness. The analysis of the adsorption isotherms showed that the R2 values matched the following order: Langmuir > Temkin > Freundlich > Dubinin-Radushkevich. The UTPS/ISC2/SICNCs showed the highest removal (%) of 83% for CV dye uptake.Corn starch was modified via the esterification process with iodine as an oxidizing agent to fabricate iodinated starch citrate (ISC). Thermoplastic starch (TPS) was manufactured using urea as a plasticizer applying the casting solution process to produce the following samples, UTPS, UTPS/ISC


With the passing of our founding Editor-in-Chief, Dr. F. Douglas Scutchfield, the Journal of Appalachian Health team reflects on a life well lived and a monumental public legacy left behind. We thank “Scutch” for enriching the lives of so many, and we commit to growing the Journal in his honor. You do not need to know precisely what is happening, or exactly where it is all going. What you need is to recognize the possibilities and challenges offered by the present moment, and to embrace them with courage, faith and hope.Conjectures of a Guilty Bystander— Thomas Merton, is. A humble recipient of health and humanities accolades alike, Scutch was not just an observer of life’s complexities but a truly active participant in shaping the systems and minds around him. He didn’t just have dreams; he “gave ’em wings” .1To say that F. Douglas Scutchfield, MD—known to many of us warmly, and quite simply, as “Scutch”—was a Renaissance man with reverberations far beyond public health seems insufficient to capture all that he was and Journal of Appalachian Health dedicated to investigating, chronicling, and advancing the well-being of the people who call Appalachia’s hills home.One of those dreams was this publication—a Scutch was himself a native of Appalachia, born amidst the Second World War in Wheelwright, Kentucky. With just over 500 residents at the last Census,This journal is Scutch’s legacy—a tour de force in a career dedicated to bettering the lives of those around him. It caps off an impressive portfolio which took Scutch from the hills of Floyd County to Chicago, Atlanta, Tuscaloosa, San Diego, Israel, and home to Kentucky again. Through his many experiences— which included practicing family medicine, founding schools of public health,Journal of Appalachian Health.As this new era for the Journal dawns, we are committed to carrying Scutch’s work forward. Dr. Scutchfield himself wrote in our last issue that there are new faces aboard to help captain the The present moment presents unparalleled challenges—both in the transition of our Journal and in society at large—but we move forward with courage and continued faith in the power of evidence-based public health research to change lives for the better. This was the task that Scutch recognized and committed himself to. Perhaps more remarkable than his mind for public health was his ability to see possibility in even the bleakest situations, and we want to capture that spirit. So as he would write, “on we go!”, with hope.


Blooms of marine microalgae play a pivotal role in global carbon cycling. Such blooms entail successive blooms of specialized clades of planktonic bacteria that collectively remineralize gigatons of algal biomass on a global scale. This biomass is largely composed of distinct polysaccharides, and the microbial decomposition of these polysaccharides is therefore a process of prime importance.β-glucans (diatom laminarin) and α-glucans as the most prominent and actively metabolized dissolved polysaccharide substrates. Both substrates were consumed throughout the bloom, with α-glucan PUL expression peaking at the beginning of the second bloom phase shortly after a peak in flagellate and the nadir in bacterial total cell counts.In 2020, we sampled a complete biphasic spring bloom in the German Bight over a 90-day period. Bacterioplankton metagenomes from 30 time points allowed reconstruction of 251 metagenome-assembled genomes (MAGs). Corresponding metatranscriptomes highlighted 50 particularly active MAGs of the most abundant clades, including many polysaccharide degraders. Saccharide measurements together with bacterial polysaccharide utilization loci (PUL) expression data identified We show that the amounts and composition of dissolved polysaccharides, in particular abundant storage polysaccharides, have a pronounced influence on the composition of abundant bacterioplankton members during phytoplankton blooms, some of which compete for similar polysaccharide niches. We hypothesize that besides the release of algal glycans, also recycling of bacterial glycans as a result of increased bacterial cell mortality can have a significant influence on bacterioplankton composition during phytoplankton blooms.Video AbstractThe online version contains supplementary material available at 10.1186/s40168-023-01517-x. The carbon cycle is the largest biogeochemical cycle. Every year, more than 100 Gt of carbon are fixed by photosynthesis in about equal amounts by terrestrial plants and marine algae . Marine Bloom-associated bacteria have co-evolved with photosynthetic microalgae since the latter emerged during the Precambrian Proterozoic period roughly two billion years ago , includiβ-1,3-linked backbone and occasional β-1,6-linked branches. Laminarin serves as storage of photoassimilated glucose in diatoms and therefore is one of the most abundant polysaccharides on Earth ).On April 1/2 and at the onset of the second phase on April 22/23, inorganic ammonium and phosphate concentrations spiked almost threefold to about 5.1–6.8 µM and 1.6–1.8 µM, respectively. Both spikes were accompanied by increases in silicate concentrations, and drops in salinities from about 33.8 to 31.6 and 32.1 to 30.4, respectively . A similar pattern was found for Planktomarina with a maximum of ~ 11% on April 30. Sulfitobacter sequences were associated with the second and terminal bloom phases and reached up to ~ 8%, while SAR116 sequences never exceeded ~ 5% at any time point.Unassembled metagenome reads contained 325,737 near full-length 16S rRNA gene sequences (average: 1269 bp) that were used for diversity analyses  were found in all bloom phases but with low frequencies. In contrast, Cd. Thioglobus (SUP05 clade) sequences were confined to the pre-bloom phase before March 26.The most prominent  in GTDB ) were alCd. Actinomarina were restricted to the pre- and first bloom phases with a maximum of ~ 5% relative abundance on March 10. Marine group II Archaea sequences were low in abundance throughout the bloom, with highest relative abundances of ~ 11% during the pre-bloom phase on March 26.Sequences of actinobacterial Aurantivirga (probe AUR452) and Polaribacter (probe POL740) as the dominant clades within the Bacteroidota, with maximum relative abundances of 18.3%  and 17.7%  on May 8 and 4, respectively . This peak coincided with the initial D. brightwellii peak, suggesting a rapid response of Aurantivirga to the proliferation of this diatom. Cd.  Abditibacter  gradually increased to a maximum of 10.7%  on May 19  probes  were more uniformly distributed, with peaks in relative abundance of around ~ 15%  on March 26/27  during the first bloom phase, and on May 8 during the second bloom phase. Abundant members comprised Amylibacter and Planktomarina, with Amylibacter being abundant throughout the bloom, and Planktomarina thriving during the second bloom phase  dominated during the pre-bloom and both major bloom phases with relative abundances ranging from 18.6%  on March 2 to 26.6%  on April 27  dominated, followed by Bacteroidota (36.3%). The remaining bacterial MAGs belonged to Actinobacteriota, Verrucomicrobiota, Planctomycetota, Marinisomatota, Myxococcota, and Campylobacterota  MAGs increased notably and peaked on March 26 during the first D. brightwellii peak with 36.3%  and 20.4%  of the TTPM, respectively. During the second bloom phase after April 27, relative activities of alphaproteobacterial MAGs decreased and reached a minimum of 18.0%  of the TTPM on May 13. Simultaneously, relative activities of Bacteroidia MAGs increased during the second bloom phase and even dominated a larger part of this phase with a maximum of 53.2%  of the TTPM on May 8  and 32.1%  of the total transcripts per million (TTPM), respectively  of the TTPM. The most active of these MAGs (AI_MB94_1) exhibited highest expression during the second bloom phase after a steep rise from 1.0% (9958) of the TTPM on April 29 to 13.7%  of the TTPM on May 8. The second-most active Aurantivirga MAG (C_MB100) exhibited maximum expression of 7.3%  of the TTPM on March 26 during the D. brightwellii peak, indicating rapid response to proliferation of this diatom. Correlation with CARD-FISH data was high , confirming Aurantivirga as prominent bloom-associated clade  of the 251 representative MAGs affiliated with Bacteroidota clade was represented by two Cd. Prosiliicoccus MAGs. Their combined maximum expression accounted for up to 9.5%  of the TTPM, with one MAG (AB_MB380) alone accounting for 7.5% . Expression of both MAGs was negligible during the pre- and first bloom phases, but started to increase after April 29 and for AB_MB380 peaked on May 6 during the second bloom phase , with a peak on May 4 with 6.0%  of the TTPM. Information on other lower expressed Bacteroidota MAGs is included in Additional file The second-most expressed MAG AB_MB80 alone .5% 95,26 of the TBacteroidota MAGs included two Cd. Abditibacter MAGs and four MAGs from different NS clades. The Cd. Abditibacter MAGs exhibited almost uniform low expression throughout March to May. In contrast, MAGs affiliating with NS5 and NS3a marine groups exhibited higher expression during the first bloom phase, while MAGs affiliating with the NS4 and NS2b marine groups had higher expression before April 27 (first phase) and after May 12   exhibited a pronounced peak in abundance and expression on May 4, making it the topmost expressed gammaproteobacterial clade with 15.4%  of the TTPM and 7.6% of relative 16S rRNA gene abundance. However, its peak expression lasted for only about a day, dropped to 2.2% afterwards, and subsequently, activity of Glaciecola Y_MB157 almost vanished with a mere 30 TPM left on May 20.Eighteen out of 78 representative gammaproteobacterial MAGs were part of the 50 topmost expressed MAGs. A single Bacteroidota clades. Two Luminiphilus (OM60/NOR5 clade) MAGs showed only little expression during the second bloom phase, with a notable increase on the last sampling date of the terminal phase  of the TTPM, suggesting a relevance akin to that of abundant In contrast, SAR86 clade MAGs showed high expression mostly during the first bloom phase, during which all four respective MAGs combined accounted for 4.0%  to 11.5%  of the TTPM. Expression decreased considerably after April 29, reaching a minimum of only 0.8%  of the TTPM on May 4. Similar to SAR92, expression of SAR86 increased again after May 8. Also, MAGs affiliating with the OM43 clade exhibited a similar overall activity pattern.Pelagibacter (SAR11 clade) MAGs . Their collective expression ranged from 4.2%  to 16.4%  of the TTPM during the pre- and first bloom phases. Lower expression was observed for the related SAR116 clade, whose expression peaked during the first bloom phase  showed high expression throughout the bloom with expression ranging from 1.7%  to 12.9%  of the TTPM. In contrast, the more active of two Planktomarina MAGs (P_MB52_1) was particularly active during the second bloom phase, with maximum expression of 10.9%  of the TTMP on April 29.The 50 topmost expressed MAGs included ten alphaproteobacterial MAGs, with highest expression of three Actinobacteriota  and Verrucomicrobiota (BACL24 clade). The top 50 expressed MAGs also contained three archaeal MAGs that all belonged to the Poseidoniaceae family and some further Gammaproteobacteria MAGs belonging to Thioglobus A (SUP05 clade) and the OM182 clade   Fig.  A, B.FigAurantivirga AI_MB94_1, Cd. Prosiliicoccus AB_MB380, Aurantivirga C_MB100, NS4 clade W_MB115, and Polaribacter P_MB288 , α-glucans (GH13), alginate (PL6-PL8-PL7), and an unspecified polysaccharide (GH3-GH130-GH18-CE2). GH0, GH16_3, and GH13 gene expression levels ranged among the top 10% in this MAG, suggesting particular active laminarin and α-glucan degradation . The first belonged to the previously defined variant 1 type laminarin PULs [Cd. Prosiliicoccus MAG among the topmost expressed MAGs, Q_MB57, had a much weaker overall expression and featured expressed variants 1 and 2 type laminarin PULs, with slightly higher expression of the latter , α-glucans (GH13-GH65-GH31), and alginate (PL6-PL7-PL17). These two MAGs exhibited an almost synchronous expression peak at the onset of the second bloom phase, indicating competition for the same polysaccharide niche, in which Aurantivirga AI_MB94_1 notably outcompeted Polaribacter P_MB288 , a putative porphyran (GH29-GH86), a sulfated α-mannose-containing polysaccharide (GH92-sulfatases), and additional unspecified glycans   were notably expressed, with all glycoside hydrolases among the top 10% of expressed genes in their respective MAGs. Laminarin-targeting Aurantivirga C_MB100 peaked in the first bloom phase, while the Aurantivirga Al_MB94_1 peaked in the second bloom with highest expression of its α-glucan-targeting PUL  and one SAR86 (P_MB137_1) MAG, sometimes with an additional GH158 , suggesting regulation.Variant 1 laminarin PULs were expressed by almost three-quarter (11/15) of the active bacteroidetal and a third (7/18) of the active gammaproteobacterial MAGs Fig. , Fig. 6.sa genus . Such PUGammaproteobacteria, Glaciecola MAG Y_MB157 possessed seven different expressed PULs, but no variant 1 laminarin PUL. Predicted targets included α-glucans (GH13-GH31), laminarin/β-glucans , alginate , and fructans (GH32)  exhibited expression of genes coding for α-L-fucosidases, arylsulfatases, and GH33 family glycoside hydrolases. Details on the predicted metabolism of Verrucomicrobiota MAGs obtained from metagenomes off Helgoland Roads were described recently in a dedicated study [In contrast to these ed study .β-1,3-glucan antibody (BS-400–2) revealed the continuous presence of laminarin  with an anti-ibody BS-00–2 reveibody BS-00–2 reveibody BS-00–2 reveibody BS-00–2 reveAntibody-based measurements also suggested an abundance of galactose-containing polysaccharides . Other abundant monosaccharides comprised, in descending order, fucose (monomer in fucoidan and other fucose-containing polysaccharides), galactose , mannose/xylose (monomers in mannans/mannose-containing and xylans/xylose-containing polysaccharides), and glucosamine  . Mild wD. brightwellii. Blooming D. brightwellii are known to inhibit proliferation of other diatom species by toxic metabolites [D. brightwellii during this phase. Size may have been another factor. D. brightwellii cells tend to form large cells, in particular in colder waters (< 15 °C) [D. brightwellii observed at Sylt Roads in the North Sea during the sampling period fell into two size classes of 40:120 µm and 70:150 µm in terms of diameter and lengths in pervalvar axis  , as presCerataulina pelagica and Chaetoceros sp. and led to a rapid depletion of silicate, ammonium, and phosphate . It is noteworthy that blooming C. pelagica have been reported to cope well with diminished silicate concentrations [Chaetoceros sp. are able to take up silicate and other nutrients at low concentrations [The second bloom phase was dominated by trations . Likewistrations  and thusBesides food web effects, also hydrodynamics at Helgoland must be considered, as influxes from distinct water bodies can influence population sizes of planktonic species. Influxes of nutrient-rich, coastal waters were detected at the beginning of the first bloom phase and in particular immediately preceding the second bloom phase. The first influx probably prolonged the first bloom phase, and the second was likely instrumental in the development of the second bloom phase. Neither physicochemical nor bacterioplankton data did suggest a fundamental change in water regime by these influx events. Nevertheless, we cannot exclude that these influxes had quantifiable effects on plankton population sizes, which precludes speculations on causes for population changes during these two well-defined events.D. brightwellii peak on March 26, bacterioplankton clades such as Aurantivirga , NS4, NS2b, Amylibacter, and SAR92 (B_MB221) showed sharp expression peaks. Likewise, Aurantivirga , Polaribacter, Cd. Prosiliicoccus (formerly classified as Ulvibacter), Glaciecola, SAR92 (except B_MB221), OM43, Ilumatobacter, and Cd. Nanopelagicales activities seemed tightly coupled to the presence of C. pelagica and Chaetoceros sp. diatoms in the second bloom phase. However, in case of the most abundant polysaccharide-degrading bacteria, the presumption of direct coupling to algae species is misleading. Laminarin PUL expression correlated well with overall diatom biomass throughout the bloom, reflecting that diatoms produce laminarin, independent of species. Likewise, the sharp increase in α-glucan PUL expression at the onset of the second phase may not have resulted from a change in diatom species composition. During this time, a steep rise in flagellate numbers was accompanied by an about threefold drop in bacterial cell counts from 1.07 × 106 per mL on April 26 down to 3.41 × 105 per mL on April 30 , OM43 (AB_MB181_1), SAR92 (B_MB221), NS4 (W_MB115), NS2b (AK_MB314_1), and UBA952 (O_MB51_1) MAGs increased. Gammaproteobacterial OM43 clade methylotrophs are frequently associated with algal blooms. They thrive on methanol and other C1 compounds that are released by algae [Pelagibacterales/SAR11, are typical oligotrophs that nonetheless can profit from an overall increased availability of nutrients . Moreovβ-glucans, α-glucans, alginates, and mannose- and xylose-containing polysaccharides. In particular, variant 1 laminarin PULs, as previously described in Formosa sp. Hel1_33_131 [We have described the diverse CAZyme and PUL repertoires of phytoplankton-associated bacterioplankton clades in previous studies e.g., , 73, 79), 7973, 7α-mannosidases in Aurantivirga C_MB100 as well as expressed laminarin and xylan PULs, whereas Aurantivirga AI_MB94_1 expressed laminarin, α-glucan, and alginate PULs. These preferences seemed to be either hardcoded or tightly co-regulated, because overall PUL expression patterns did not change in most Aurantivirga MAGs. An exception was Aurantivirga MAG C_MB344 that showed a notable downregulation of its α-glucan PUL relative to its laminarin PUL during the second bloom phase  and 3 Mbp, except Luminiphilus T_MB10, OM182 P_MB234_1, and Ilumatobacter C_MB28, whose MAGs ranged up to ~ 3.5 Mbp. The highest expressed MAG during the second bloom phase, Aurantivirga AI_MB94_1 and Cd. Prosiliicoccus AB_MB380, had sizes around 2.0 to 2.2 Mbp. Regulation is among those traits that are typically reduced during genome streamlining — Pelagibacter ubique being a prime example [Bacteroidota for a swift response to bloom situations and might be among the reasons why Bacteroidota usually outcompete Gammaproteobacteria at the onset of spring phytoplankton blooms at Helgoland Roads ..PelagibaFormosa strains [Reinekea strain [Cd. Prosiliicoccus vernus from bloom-associated metagenome data [During the last decade, we have isolated thousands of North Sea bacterial strains and classified them using 16S rRNA gene sequencing , 31. Bas strains  and one a strain , 30 thatome data . Still, α-glucans, play a pivotal role in this process. Also, alginates seem to be readily metabolized, which not only constitute a cell wall component of brown algae but are also produced by some bloom-associated bacteria [Microbial decomposition of algal biomass is a concerted effort of both bacterial specialists and generalists with distinct substrate niches. While resource partitioning is particularly evident for polysaccharides , it alsbacteria . HoweverSamples were taken from March to May 2020 at the long-term ecological research site “Kabeltonne”  off the coast of Helgoland Roads (North Sea) during a diatom-dominated spring phytoplankton bloom. Seawater was sampled at 1 m depth five times a week as described previously  for a toTen liters of seawater were filtered sequentially through 10, 3, and 0.2 µm pore-size polycarbonate filters (142 mm diameter) for bacterial biomass. The 10 and 3 µm filters retained most of the eukaryotes, larger particles, and attached bacteria, while the bulk of the free-living bacteria were collected on the 0.2 µm filters. All filtrations were performed in duplicates, and filters were immediately flash frozen in liquid nitrogen and kept at − 80 °C until further use.For cell counting, 10 or 100 mL of unfiltered seawater was fixed with 37% formaldehyde (v/v) to a concentration of 1% (v/v) for 1 h at room temperature. The fixed samples were then filtered directly onto 0.2 µm pore-sized polycarbonate filters (47 mm diameter). All filters were preserved at − 80 °C until further use.Aurantivirga), POL740 (Polaribacter), Vis6-814 (Vis6 clade including Cd. Abditibacter), SAR86-1245 (SAR86 clade), SAR92-627 (SAR92 clade), OM182-707 (OM182 clade), SAR11-mix (SAR11 clade including Pelagibacter), and Ros537 (Roseobacter clade). Corresponding sequences are provided in Additional file Bacterial cells were stained with DAPI  and counted microscopically for total cell number estimates. Likewise, cells of prominent clades were stained with specific probes for CARD-FISH and counted microscopically. Both techniques were applied as described previously . CARD-FIWind direction data were obtained from the Climate Data Store of the Copernicus Climate Change Service . All otha data and cell numbers, we selected 27 time points for sequencing. On three dates, samples were taken twice a day , in order to possibly test expression with and without sunlight (not part of this study). This amounts to a total of 30 metagenomes and corresponding metatranscriptomes. Sampling dates and further details are provided in Additional file Metagenome and metatranscriptome sequencing of biomass from the 0.2 µm filters were performed at the Max Planck Genome Centre . For each time point, one filter was used for the extraction of both DNA and RNA. Isolated RNA was processed to deplete ribosomal RNAs using oligo-based customized probes , and themeta and − pacbio-hifi. 16S rRNA sequences were extracted from metagenome assemblies using Barrnap v0.9 [Metagenome sequencing was performed on a PacBio Sequel II  using one SMRT cell per sample in long-read HiFi mode. Details on the raw data are provided in Additional file nap v0.9  and clasnap v0.9 , 61. A sReconstruction of MAGs (metagenome-assembled genomes) was carried out in Anvi’o v7.0 . Initialbbduk and repair scripts of the BBMap v35.14 suite (https://sourceforge.net/projects/bbmap/). Reads with a minimum read length of 70 bp were subsequently mapped onto all 251 representative MAGs using Bowtie2 [Metatranscriptome sequencing was performed on an Illumina HiSeq 3000 . About 100 million paired-end reads (2 × 150 bp) were generated per sample. Ribosomal RNA reads were filtered using SortMeRNA v3.0 . Remaini Bowtie2  as a parw1 and sanger_5) [usCD, or susC genes as PULs and co-localizations of CAZyme genes without susCD or with other transporters  as CAZyme clusters. Mapping of mRNA reads against contigs (concatenated MAGs) was performed using Bowtie2, and transcripts per million (TPM) values were calculated for all MAGs of a given sample as follows: (Σ reads of sample successfully mapping to a MAG × 10^6)/(Σ lengths of contigs of the MAG × Σ number of reads in the sample). Results were visualized using the SQMtools R package.We used SqueezeMeta in merged mode for the integrated analysis of metagenomes and corresponding metatranscriptomes. Concatenated MAGs were supplied to SqueezeMeta as metagenome assembly. Contig statistics were calculated using Prinseq v0.20.4 . RNAs weanger_5)  and searanger_5) , eggNOG anger_5) , KEGG r5anger_5) , and CAZanger_5)  using Dianger_5) . HMM homanger_5)  against anger_5) . CombineHigh molecular weight DOM (HMWDOM) was sampled using tangential flow filtration in parallel to OMICs sampling from the same water body. Samples were processed as described previously  with sliAliquots of the HMWDOM samples were also used for monosaccharide analysis by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) with a Dionex CarboPac PA10 column  as described elsewhere , 75.Additional file 1: Fig. S1. A. Levels of salinity and temperature. B. Total bacterial cell counts and flagellate counts. C, D. Concentrations of  silicate, phosphate, nitrate, and ammonium. Fig. S2. Wind directions and speeds at 10 m above the sea surface level as obtained from the Climate Data Store of the Copernicus Climate Change Service (ERA 5 product). Corresponding wind vector data is provided in Additional file 4. Fig. S3. Quality measures of the MAGs obtained in this study. Fig. S4. Overall expression of all mentioned clades in terms of transcripts per million during the 2020 North Sea spring bloom. Fig. S5. Transcription pattern of seven archaeal MAGs obtained in this study. Fig. S6. Transcriptional profiles of PULs and their predicted polysaccharide substrates in Aurantivirga MAG AB_MB169 and C_MB344. Fig. S7. Transcriptional profiles of PULs and their predicted polysaccharide substrates in Cd. Prosiliicoccus MAG Q_MB57 and Polaribacter MAG X_MB288. Fig. S8. Transcriptional profiles of PULs and their predicted polysaccharide substrates in Abditibacter MAG D_MB74 and L_MB280. Fig. S9. Transcriptional profiles of PULs and their predicted polysaccharide substrates in NS4 clade MAG W_MB115. Fig. S10. Transcriptional profiles of PULs and their predicted polysaccharide substrates in all SAR92 clade MAGs: AK_MB88_1, B_MB221, X_MB111, V_MB374, AB_MB236 and SAR86 MAG: P_MB137_1. Fig. S11. Transcriptional profiles of PULs and their predicted polysaccharide substrates in Luminiphilus OM60 (NOR5) clade MAGs: AA_MB219 and T_MB10. Fig. S12. Transcriptional profiles of PULs and their predicted polysaccharide substrates in the highly expressed and sole Glaciecola MAG Y_MB157. Fig. S13. Antibody-based measurements (1→4)-β-D-galactan) of galactose-containing polysaccharides extracted either with MilliQ or EDTA (lines) as compared to the estimated algal biomass data (gray area). Fig. S14. Comparison of the combined expression of PULs targeting laminarin and α-glucan storage polysaccharides that both consist entirely of glucose, and the measured concentrations of glucose in polysaccharides from HMWDOM. Fig. S15. Measured concentrations of monosaccharides other than glucose in HMWDOM across samples.Additional file 2: Total algal cell counts and biovolumes (BV), inorganic nutrients and precipitation measured during the 2020 Helgoland spring phytoplankton bloom.Additional file 3: Copepod counts during the 2020 Helgoland spring phytoplankton bloom.Additional file 4: Wind vector components, direction and speed data at 10 m above the sea surface. Additional file 5: Sampling dates and statistics of 16S rRNA sequences extracted from PacBio Sequel II raw reads.Additional file 6: Relative abundances of 16S rRNA gene sequences extracted from metagenomes.Additional file 7: Microscopic cell counts with fluorescently labeled CARD-FISH probes.Additional file 8: Numbers of bins obtained by metagenome binning using Anvi'o.Additional file 9: GTDB affiliation of all MAGs dereplicated at 0.95 ANI along with other genomic features.Additional file 10: TPM (transcripts per million) values of major clades during the 2020 Helgoland spring phytoplankton bloom.Additional file 11: Monosaccharide concentrations resulting from acid hydrolysis of high molecular weight DOM polysaccharides.Additioanl file 12: Antibody-based  measurements of dissolved laminarin extracted either with MilliQ or EDTA.Additional file 13: CARD-FISH probes used in this study.Additional file 14: Sampling dates and statistics of raw data obtained for metagenomes and metatranscriptomes.Additional file 15: Polysaccharide measurements during the 2020 Helgoland Spring phytoplankton bloom.