Betula pendula Roth.) for coating of aspen wood (Populus tremula L.). The SFA combined with maleic anhydride (MA) and octadecyltrichlorosilane (OTS) as a curing agent was prepared in ethanol and used in surface coating. The water contact angle, surface reflectance spectra, FTIR, and SEM-EDS were used to characterize the physical and chemical properties of the coated wood samples. Further, the long-term stability of the SFA coating was analyzed via artificial aging. The wood surface became hydrophobic, as the contact angle for the water droplet (WCA) was over ~120°, and was stable for all of the prepared combinations of SFA, MA, and OTS.Bark extracts are sustainable sources of biopolymers and have great potential to replace fossil-based polymers in wood coating applications. The present study investigated the applicability of suberin fatty acids hydrolysate (SFA) extracted from the outer bark of silver birch ( As a hydrophilic, hygroscopic, porous, and fibrous material, wood is especially vulnerable to water sorption, because water penetrates rapidly into the wood structure causing swelling and eventually a loss of mechanical strength as well as providing conditions for biological degradation ,2. Sever3 [Betula pendula Roth.) is made up of 45% suberin, 40% extractives, 9% lignin, 4% hemicelluloses, and 2% cellulose, on average [The wood processing industry provides large amounts of bark as a side stream, and most of it is incinerated for energy instead of cascading for potential green chemicals ,9,10). T3 . In birc average . The ext average ,14. Birc average ,15. The  average . Birch b average . Suberin average ,18, a so average , a therm average , a water average , and as  average  evaluateIn this study, the suberin fatty acids hydrolysate (SFA) of outer birch bark was investigated as a potential hydrophobic coating application on aspen wood surface. Two crosslinking agents, maleic anhydride (MA) and octadecyltrichlorosilane (OTS), were used in several combinations. The coated wood samples were evaluated by Fourier transform infrared spectroscopy (FTIR), surface reflectance spectra, and scanning electron microscopy with energy-dispersive X-ray analysis (SEM-EDX). In addition, the water contact angle was measured using the sessile drop method. Further, the effectiveness of the hydrophobic properties of wood samples was evaluated by exposing them to artificial aging.Betula pendula Roth.) was manually removed from freshly cut stems (tree diameter: 200–300 mm), followed by air-drying at room temperature. The outer bark was then ground using a cutting mill Fritsch PULVERISETTE Type 15.903  with a sieve cassette having 4 × 4 mm2 square openings. The ground outer bark was then freeze-dried and stored in an airtight polyethylene bag.The outer bark of silver birch (Populus tremula L.) was procured from a local sawmill in southern Finland and prepared as specimens free from knots and cracks with straight grains and normal growth ring widths. The wood samples were prepared to uniform dimensions (100 × 45 × 5 mm) and oven-dried at 105 °C for 24 h prior to coating.European aspen wood , sodium hydroxide 99.0% , ethanol 94.0% , and sulfuric acid 95%  were used in the experiments. Then, 2 molar sulfuric acid was prepared by pouring 112 mL of concentrated sulfuric acid into 600 mL of maxima ultrapure water. The final volume was adjusted with ultra-pure water to 1000 mL after the solution was cooled to room temperature.v/v). On o.d. bark, 20% NaOH was applied, and the bark was boiled in the solution for 3 h. The betulinol fraction and suberin fatty acids were then separated. The extractions were repeated three times. The different fractions were characterized separately and mixed before the wood surface treatment.The dry solid content of the ground and dried outer bark was 97.8%. The hydrolysis and extraction were carried out according to a protocol described by Korpinen et al. (2019) . In the C). SFA–MA): Fixed amounts (70:30) of SFA and maleic anhydride as a hardener were mixed and boiled at 45 °C in ethanol. A manual brush coating was used to prepare a uniform two-layer coating of SFA–MA on wood and dried for 24 h at 105 °C;SFA and maleic anhydride (MA) : SFA–MA-coated samples dipped into a solution (1/100) (v/v) of OTS and n-hexane for 30 min and dried for 24 h at 105 °C;Treatment of coated samples with octadecyltrichlorosilane (OTS)  (v/v) of OTS and n-hexane for 30 min and dried at 24 h at 105 °C;SFA–OTS: In 100 mL ethanol, 5 g SFA and 500 µL OTS were mixed and boiled at 120 °C (10 min) and cooled down to room temperature and applied manually to the wood surface (two coatings).The wood samples without any coating were selected as a control trifluoroacetamide (BSTFA):chlorotrimethylsilane (TMCS):pyridine (120:20:20) and heated for 50 min at 70 °C. The silylated samples were quantified by gas chromatography flame ionization detection (GC-FID), and the peak identities were confirmed by gas chromatography mass spectrometry (GC-MS) as described by Korpinen et al. (2019) .−5 (high vacuum) and acceleration voltages of 15 and 10 kV were used to measure the specimen’s surface. Further, energy-dispersive X-ray spectroscopy (EDS) was applied with ESPRIT v.2.1.2.17832 coupled with SEM.The surfaces of coated wood samples and uncoated reference samples were evaluated using scanning electron microscopy . The specimens were coated with a thin evaporated layer of carbon to improve conductivity prior to the analysis. The prepared specimens were mounted 12 mm from lenses. The chamber pressure < 1.0 × 10The surface reflectance spectra of coated wood samples and uncoated reference samples were measured at intervals of 8 mm in diameter for each specimen in the visible light wavelength range of 360–740 nm using a Konica Minolta CM-2600d spectrophotometer . Spectral data were converted to CIEL*a*b* color coordinates using a 2° standard observer and a D65 light source for lightness (L*), redness (a*), and yellowness (b*), according to the CIEL*a*b* color space (ISO 11664-4:2008). For each sample group, the mean and standard deviations of the color coordinates were calculated.p < 0.05 levels for the color differences in samples before and after artificial aging.One-way ANOVA was performed using the Origin  statistical data analysis software to calculate the samples’ mean difference at The wettability of the coated wood samples and uncoated reference samples was evaluated based on the water contact angle (WCA) using the sessile drop method. The contact angles were measured by means of computer-aided analysis  of elliptical shapes of liquid drops as observed in an optical goniometer and recorded by a digital camera installed in axial extension of the lens and statistics of 5 droplets per specimen. Drops of approximately 4–6 μL, the volumes being calculated from images of the drops, were applied by means of a dispenser.−1 with a scanning rate of 2 cm−1 and 50 scans per run.Fourier transform infrared spectrophotometer  coupled with IRsolution software were used to gain, control, and process the data. Semi-thin layers were cut from wood specimens with a razor blade and then dried at 60 °C for two hours. The prepared specimens were scanned while using an attenuated total reflection (ATR) setup in the absorbance range of 400–4000 cm−2 in the range of 300–400 nm at 65 °C was used (black standard).The artificial aging of coated wood samples and uncoated reference samples were performed to compare and understand the long-term performance of prepared coating formulations. The aged samples were characterized to see the changes due to the fact of aging. The artificial aging was carried out according to ISO 4892-2:2013 using an Atlas Xenotest 440 weather testing chamber  for 500 h. Xenon light exposure at 60 Wm−1 as shown in −1 of the carbonyl groups of typical esters was present in suberin. The symmetric and asymmetric bonds associated with vinyl groups, such as C–O and C–H, were present at 1245, 1164, and 722 cm−1. The characteristic epoxy band was present at 845 cm−1 of hydroxyl-fatty acids, i.e., 9,10-epoxy-18-hydroxy-18:0 acid [−1 [Two integral and characteristic peaks of a long aliphatic chain in suberin appeared at 2919 and 2851 cm8:0 acid ,24, whic8:0 acid . The banThe chemical composition of the suberin fatty acids in the hydrolysate can be seen in −1 of the O–H group stretching vibration from the intermolecular and intramolecular hydrogen bonds [−1 in the methyl and methylene groups [−1 showed the absorption bands of functional groups present such as C–O at 1732 and C–C at 1505 cm−1 [−1, etc. [−1 for terminal methyl groups with different intensities and Si–O–C at 1192 cm−1. The OTS treatment usually deposited the hydrophobic self-assembled monolayers via a hydrolysis and condensation process [The infrared spectra of uncoated reference aspen wood (Sample C) showed absorption bands at 3345 cmen bonds  in Figure groups  and the 505 cm−1 . Several−1, etc. . The OTS process .−1 associated to cellulose C6–O6H C–O stretching in lignin shifted to a lower absorbance intensity, and a major shift appeared in the SFA–OTS combination. Similar to this, several other functional groups ; 1462 cm−1 (lignin C–H bending); 1736 cm−1 (C=O stretching); aliphatic chain (CH3 and CH3 at 2919 and 2851 cm−1) were more visible in the SFA–OTS coating system. The peak at 1248 cm−1 associated with C–O stretching in carboxylic acid shifted to a lower absorbance intensity after all the different coatings, possibly due to the bonding with reactive moieties of SFA and crosslinking agents. Another important characteristic peak belonged to SFA’s ester group at 1736 cm−1, and hemicellulose (C=O) significantly shifted to a higher peak absorbance for the SFA–OTS coating system, while it did not change for other combinations of coatings. Importantly, the OH stretching at ~3350 cm−1 also changed after SFA-based coating systems.In this study, two crosslinking agents were used to crosslink the hydroxy groups present in the SFA hydroxylates. At first, MA was used as a crosslinking agent, and the SFA underwent a maleation reaction due the reaction with MA and maleated SFA having two-way interactions with hydroxy groups; it reacted with epoxides first and the remaining hydroxy groups later . After SThe SEM images of the longitudinal section of the samples are presented in −1 that the OTS hydrolyzed first and reacted with free –OH groups  was 60 ± 10°, and it was reduced to 17° after 7 s from when the water droplet was absorbed onto the wood . The WCAIn the color measurement, the uncoated reference (sample C) averaged 89 ± 2 for lightness, 2 ± 1 for redness, and 21 ± 2 for yellowness . After O−1 . The intensity of all these bands and peaks almost diminished due to the exposure to UV light [−1 of syringyl ring vibration and the C–O stretch of lignin and xylan also disappeared due to the fact of aging under xenon light. On the other hand, the change in the polysaccharides’ absorption bands at 1157 (C–O–C vibration in cellulose and hemicellulose), 1107, and 1052 cm−1 in comparison to the unexposed sample [Comparison of FTIR spectra of unexposed and aged wood surfaces with different coatings are shown in UV light . The vibd sample .−1 in the methyl and methylene groups disappeared, and the intensity vibration peak at 1268 cm−1 increased significantly, the results being associated with the presence of a syringyl ring. The peak associated to the carbonyl group at approximately 3400 cm−1 showed an increased intensity due to the fact of aging for the uncoated refence (sample C), OTS samples, and SFA–MA samples. The samples being coated with combinations, including SFA and OTS, showed high stability in terms of surface chemistry alteration due to the fact of aging.In the case of the coated samples, significant changes after aging were observed in the SFA–MA samples. The peaks associated to C–H stretching absorption between 2850 and 2930 cmUsually, most of the protection coating on wood surfaces deteriorates with time due to the continuous change in external environmental conditions; therefore, the water contact angle was measured after the artificial aging of wood surfaces . The WCAAs can be seen in In this study, the suberin fatty acid hydrolysates (SBF) extracted from outer birch bark with a majority composition of 9,10-epoxy-18-hydroxy-18:0 acid was investigated as bio-based polymeric materials for wood coating applications. SFA alone was not able to form any bonding with the wood surface; thus, two types of crosslinking agents, MA and OTS, were used. Different combinations of SFA with OTS and MA were used for wood coating, i.e., OTS alone, SFA–OTS, SFA–MA, and SFA–MA + OTS. The physico-chemical analysis revealed that all SFA-based coating combinations significantly improved the wood surface’s hydrophobicity. The use of OTS as a crosslinking agent for SFA was investigated for the very first time in this study. Even a layer of OTS on the SFA–MA coat contributed to the hydrophobicity of the coating. The color of the wood surface changed notably with the different coatings. Further, the long-term stability of coated wood samples was evaluated using artificial aging up to 500 h in a xenon weather testing chamber, and the aged samples were evaluated for physico-chemical properties. The results showed that all of the coating combinations were stable. The color changes were most notable in the SFA-based coatings: the lightness increased and, especially, the yellowness, but also the redness decreased. Yellowness decreased also in the OTS-coated and uncoated woods, making the wood lighter.In terms of crosslinker impact on an SFA-coating formulation, MA is mostly used in bio-based polyester crosslinking and is relatively successful in various application. OTS was used for the very first time as a crosslinker for SFA, and it performed better than MA. However, the combination of both MA and OTS performed better in comparison to all combinations. The extraction process of suberin fatty acids from birch bark could affect the quality of bio-based polyesters. Thus, important considerations need to be taken into account during the extraction process in the future course of research work. In conclusion, suberin fatty acid hydrolysates from birch bark could be potential bio-based, renewable substitutes for fossil-based polymeric materials to be used in wood coating.


Vibrio vulnificus is a naturally occurring, potentially lethal pathogen found in coastal waters, fish, and shellfish. Sewage spills in coastal waters occur when infrastructure fails due to severe storms or age, and may affect bacterial populations by altering nutrient levels. This study investigated effects of sewage on clonal and natural V. vulnificus populations in microcosms. Addition of 1% sewage to estuarine water caused the density of a pure culture of V. vulnificus CMCP6 and a natural V. vulnificus population to increase significantly, by two to three orders of magnitude, whether measured by quantitative PCR (qPCR) or culture and in batch and continuous cultures. Changes in the transcription of six virulence- and survival-associated genes in response to sewage were assessed using continuous culture. Exposure to sewage affected transcription of genes that may be associated with virulence, i.e., it modulated the oxidative stress response by altering superoxide dismutase transcription, significantly increasing sodB transcription while repressing sodA. Sewage also repressed transcription of nptA, which encodes a sodium-phosphate cotransporter. Sewage had no effect on sodC transcription or the putative virulence-associated genes hupA or wza. The effects of environmentally relevant levels of sewage on V. vulnificus populations and gene transcription suggest that sewage spills that impact warm coastal waters could lead to an increased risk of V. vulnificus infections.IMPORTANCEVibrio vulnificus infections have profound impacts such as limb amputation and death for individuals with predisposing conditions. The warming climate is contributing to rising V. vulnificus prevalence in waters that were previously too cold to support high levels of the pathogen. Climate change is also expected to increase precipitation in many regions, which puts more pressure on wastewater infrastructure and will result in more frequent sewage spills. The finding that 1% wastewater in estuarine water leads to 100 to over 1,000-fold greater V. vulnificus concentrations suggests that human exposure to oysters and estuarine water could have greater health impacts in the future. Further, wastewater had a significant effect on gene transcription and has the potential to affect virulence during the initial environment-to-host transition. Vibrio vulnificus  can also proliferate following these events (V. parahaemolyticus levels with the amount of wastewater treatment plant (WWTP) effluent released into Narragansett Bay, Rhode Island  and clinically associated (16S rRNA B or vcgC) genotypes. The 16S rRNA A/B and vcgC/E typing methods are both used frequently and have a high degree of concordance  can promote survival and virulence under acid stress and phagocyte engulfment in V. vulnificus, V. alginolyticus, and Salmonella enterica (46–wza) increases survival of V. vulnificus in the presence of serum  determine if sewage can serve as a nutrient source for autochthonous V. vulnificus populations; and (ii) determine if sewage alters the transcription of virulence- and survival-associated genes.Sewage could directly influence the probability of human infection by V. vulnificus population. In a pilot study, we employed a bioreactor in a flow-through configuration with non-sterile natural estuarine water with or without 1% sterile sewage. The density of V. vulnificus was monitored daily by qPCR of vvhA. After 24 h, V. vulnificus levels increased over 2 orders of magnitude to 8.93 × 106 GC/100 mL in the sewage treatment compared with 8.11 × 104 GC/100 mL in the control reactor  and sterile estuarine water were selected to culture V. vulnificus CMCP6. V. vulnificus density in microcosms containing nutrient replete MM9 (4.67 × 109 GC/100 mL) was not significantly different from MM9 with 1% added sewage (5.47 × 109 GC/100 mL) or from cultures grown in undiluted sewage (5.33 × 109 GC/100 mL)  amended with 1% sewage (NPC lim + 1% Sew) reached a density of 6.81 × 107 GC/100 mL, while V. vulnificus concentrations in NPC-depleted medium without sewage were below the limit of detection (<10 GC/mL) (data not shown). The addition of 1% sterile sewage to sterile estuarine water caused a significant 1.16 log10 GC/100 mL increase in V. vulnificus density to 4.21 × 107 GC/100 mL compared with the sterile estuarine water (2.88 × 106 GC/100 mL) (After observing that sewage could support the growth of autochthonous /100 mL) . V. vuln/100 mL) .V. vulnificus to assess the response of a natural population to sewage. Autochthonous populations in natural estuarine water were incubated in batch cultures ± 1% sterile sewage for 24 h . Sewage exposure significantly increased Fe SOD (sodB) transcription 2.7-fold over the control  were not significant. hupA transcription was diminished in the presence of sewage, but the change was significant only at α = 0.10 (P = 0.08).The possibility that compounds in sewage could affect the transcription of virulence- and survival-associated genes was tested using  control . ConversGlobally, 48% of untreated sewage is discharged into the environment primarily from developing countries . Contami55–Vibrio spp. concentrations following storm events have been attributed to reduced salinity and mixing of shallow and deep waters  are largely unexplored and may represent a threat to human health, as higher concentrations of pathogenic Vibrio spp. significantly increase the risk of infections  is 110 to 350 mg/L . The concentration of 1% sewage used here was selected as it represents a reasonable level of contamination following a recent, local sewage spill or chronic contamination. We base this assessment from a review of human-associated Bacteroides genetic marker (HF183) which is commonly used to identify sewage contamination of surface waters (5 to 6.15 × 106 GC/100 mL have been measured in sewage diluted 100-fold (1%)  –65 whichaters 65–.A low level of organic carbon (3.0 mg/L) was tested to simulate the level of organic carbon from primary production in an estuary (mean 3.07 mg/L)  but did V. vulnificus CMCP6 and natural V. vulnificus populations. Growth of V. vulnificus CMCP6 in sterile estuarine water without sewage resulted in a population density of ∼106 GC/100 mL measured by qPCR, which is at the upper level of previous reports from Gulf of Mexico coastal waters  were similar to previously observed levels assessed by MPN  transcription increased 2.48-fold in iron replete versus depleted media (sodB (Fe SOD) transcription and sodA (Mn SOD) repression observed here is consistent with fur-mediated gene regulation in the presence of iron previously observed in V. vulnificus and Escherichia coli . Fe SOD expression has been shown to be more important for virulence expression in mice than either sodC (CuZn SOD) or sodA  than th 31.6 μM ) indicatd effect . nptA ensporters . While tnditions  may supp. aureus , by enabV. vulnificus population in multiple ways by stimulating growth and increasing the transcription of multiple virulence associated genes. Due to the limitations of qPCR, only a small number of genes could be tested and the biological significance of these changes remain unclear. Incorporation of transcriptomic analyses and virulence phenotyping experiments would enable better associations between sewage exposure and human health risks. The response of V. vulnificus and other pathogenic Vibrio species to sewage may also enable better modeling of human health risks. Studies comparing opportunistic pathogens to obligate pathogens will be important to understand the broader impacts of sewage on waterborne pathogens and risk to human health.Sewage contamination of surface waters represents a direct threat to human health through exposure to human pathogens. Detection of fecal contamination using standard methods can take up to 24 h to 48 h and relies on identification of sewage discharge. This can put public health at risk during the interim; or worse, the discharge goes undetected and can result in an outbreak . This sV. vulnificus strain CMCP6 was maintained on Luria-Bertani agar (Difco). V. vulnificus CMCP6 broth cultures prepared for inocula in microcosm and gene transcription experiments were incubated for 20 h to 24 h in Luria-Bertani (LB) broth at room temperature (22°C).Untreated sewage (primary influent) was collected from Falkenburg Advanced Wastewater Treatment Plant, Tampa, Florida, transported on ice, and held for a maximum of 2 h before being frozen at −20°C. Sewage was held in the freezer for a maximum of 1 month prior to thawing and filter sterilization with a Rexeed 25-S hollow-fiber filter (Asahi Kasei). Three 0.1-mL aliquots of filtered sewage were spread on 100 mm Trypticase soy agar plates to check sterility, and were consistently negative for growth of colonies. Estuarine water was collected from Ben T. Davis Beach (BTD) Tampa, Florida, 27°58’12.9’’N, 82°34’42.9’’W  and Hudson Beach, Hudson Florida, 28°21'46.3“N 82°42'33.6”W  and used to construct microcosms, or sterilized and frozen, within 4 h of collection.V. vulnificus, measured at 3.23 × 103 GC/100 mL by qPCR of the vvhA gene, was collected at BTD and used to fill the 1-L bioreactor and 10-L reservoir . The sewage treatment was amended with 1% (vol/vol) sterile sewage and 3.0 mg/L glucose while the control culture received 1% sterile estuarine water and 3.0 mg/L (16.7 μM) glucose. The 3.0 mg/L glucose supplement was included to mimic natural levels of organic carbon found in estuarine water (V. vulnificus was monitored daily by filtering 50 mL of the culture through a 0.45 μm nitrocellulose filter. Filters were stored at −80°C until DNA extraction was performed using a Power Water DNA Extraction Kit (Qiagen) followed by qPCR targeting vvhA ..V. vulniV. vulnificus CMCP6 in microcosms with and without sewage. V. vulnificus concentrations were measured by qPCR of the vvhA gene  (1 CFU/mL and incubated at 37°C with shaking at 150 rpm for ∼22 h.The ability of sewage to serve as a nutrient source was assessed by incubating vhA gene  75). Al. AlV. vu(pH 7.4) . A 100 μV. vulnificus CMCP6 growth were investigated by preparing defined medium lacking each macronutrient. Media were amended with sterile sewage to serve as the sole source of the missing nutrient to determine their effects on culture density. Control (nutrient-replete) microcosms contained 20 mL of modified M9 minimal (MM9) media consisting of 50 mM tris HCl (pH 7.5), 10 mM NH4Cl, 0.1 mM CaCl2, 1 mM MgSO4, 1 mM KH2PO4, 0.1 mM ferric citrate (C6H5FeO7), 10 ‰ NaCl, and 11.1 mM (2 g/L) glucose. Casamino acids and yeast extract were omitted from MM9 to limit sources of nitrogen, phosphorous, and carbon. A medium depleted of nitrogen, phosphorous, and carbon was prepared by omitting NH4Cl, KH2PO4, and glucose. Estuarine water from Hudson Beach, Florida  was sterilized using hollow-fiber filtration (Rexeed 25-S) for microcosms made with environmental water. Sewage-amended treatments received 1% (vol/vol) sterile sewage influent. An undiluted sterile sewage treatment was amended with NaCl to a salinity of 10%. Following incubation, 20 mL of culture, or 1 mL for high cell densities, were filtered through a 0.45-μm nitrocellulose filter to concentrate bacteria. Membrane filters were stored at −80°C until DNA could be extracted using a DNeasy Power Water kit (Qiagen) and V. vulnificus was quantified using qPCR of the vvhA gene.Effects of the macronutrients nitrogen, phosphorous, and organic carbon in sewage on V. vulnificus populations in estuarine water were assessed in batch cultures. Microcosms (500 mL) were constructed in triplicate using estuarine water from BTD . We used a control treatment , a low-level glucose amendment (3.0 mg/L glucose) according to the FDA Bacteriological Analytical Manual standard method (The effects of nutrient amendment on culturable concentrations of autochthonous  glucose), and a sd method . Plates hupA, nptA, sodA-C, tufA, and wza) by V. vulnificus CMCP6 in response to sewage were assessed using an Infors-HT II bioreactor in a chemostat (continuous flow) configuration. Genes were selected based on their potential dual roles in survival in the environment and the human host, or known importance for virulence expression. Defined minimal medium containing 23.3 mM Na2HPO4, 11 mM KH2PO4, 9.35 mM NH4Cl, 85.6 mM NaCl, 1 mM MgSO4, and 2.25 mM glucose (0.405 g/L) with pH adjusted to 7.5 was used as a growth medium. The 1-L culture vessel and 4-L reservoir were filled with medium and the bioreactor was set to pH 7.5, temperature 37°C, dissolved oxygen >70%, 150 rpm stir rate, and a flow rate of 3 L/day. The V. vulnificus CMCP6 inoculum was grown at room temperature for ∼22 h in LB and 1 mL of culture was added to the bioreactor to reach a starting concentration of 106 CFU/mL. After inoculation, the bioreactor was run continuously for 48 h prior to sampling under control (no sewage added) conditions. Sampling under control conditions occurred thrice over the course of 4 h. After sampling under control conditions, the nutrient reservoir was replaced with minimal medium amended with 1% (vol/vol) sterile sewage and allowed to run for another 48 h. This allowed for the same culture sampled for the control to be exposed to the sewage treatment. After 48 h, the sewage treatment was sampled thrice over the course of 4 h.Changes in transcription of six virulence- and survival-associated genes  followed by a DNase treatment using a TURBO DNA-free Kit (Invitrogen). Briefly, RNA was diluted to 10 ng/μL and used for reverse transcriptase qPCR (RT-qPCR) of the following genes:  and wza . Thermo  and wza , 2 μL ofce gene,  with tufnce gene .Statistical analyses on culturable bacterial concentrations, qPCR, and RT-qPCR data were performed in R v3.6.3 and GraphPad Prism v8. ANOVA followed by Tukey’s honest significance tests was performed using GraphPad and the package multcomp in R.


Nonalcoholic fatty liver disease (NAFLD) is characterized by lipotoxicity and ectopic lipid deposition within hepatocytes. Sulforaphane (SFA), an active compound used for inhibiting tumors, was found to have the potency to improve lipid metabolism. However, its molecular mechanisms on ameliorating NAFLD are still incompletely understood. This research evaluated if SFA could inhibit hepatic steatosis and apoptosis. The effects of SFA on cell viability, lipid accumulation, triglyceride (TG) contents, apoptosis, ceramide contents, and reactive oxygen species (ROS) levels were analyzed in palmitic acid (PA)-treated HepG2 cells and high-fat diet (HFD)-fed mice. The related molecular mechanisms were further explored in hepatocytes. The results showed SFA alleviated lipid accumulation and regulated AMPK/SREBP1c/FAS signaling pathway in PA-stressed HepG2 cells. In addition, SFA alleviated PA-mediated apoptosis, downregulated the expressions of cleaved caspase 3, as well as reduced ceramide contents and ROS levels. Moreover, SFA treatment reduced HFD-induced body weight gain, alleviated insulin resistance, decreased serum TG, total cholesterol (TC), and alanine aminotransferase (ALT) levels, and prevented lipid deposition and apoptosis in the liver. This study showed SFA suppressed lipid deposition and apoptosis both in vitro and in vivo, indicating that SFA may be a potential candidate for preventing and treating NAFLD. Nonalcoholic fatty liver disease (NAFLD) is characterized by the excessive lipid deposition in hepatic cells (more than 5% fat contents in the liver) and describes a spectrum of complicated pathological processes, such as nonalcoholic simple fatty liver, nonalcoholic steatohepatitis, liver fibrosis, and even predisposes one to liver-related complications, such as cirrhosis and hepatocellular carcinoma . NAFLD iThough the mechanisms are still unclear, a “two hits” model has been proposed for the pathogenesis of NAFLD. Hepatic excess fat accumulation is the “first hit”, which is a progression of triglyceride accumulation induced by the imbalance between the import and synthesis of hepatic lipids on the one hand, and β-oxidation and outflux on the other . The “seSulforaphane (SFA), an isothiocyanate found in cruciferous vegetables, was firstly used for inhibiting tumors ,13. ReceSFA (purity > 99%) was obtained from LKT Laboratories . Human recombinant insulin and palmitic acid (PA) were purchased from Sigma-Aldrich, USA. Annexin V-FITC apoptosis detection kit was from Life Technologies . BCA protein concentration determination kit, total cholesterol (TC) assay kit, triglycerides (TG) assay kit, aspartate aminotransferase (AST) assay kit, and alanine aminotransferase (ALT) assay kit were from Nanjing Jiancheng Bioengineering Institute, China. All chemicals used in the research were of analytical grade.2 and 95% humidity. Human hepatic carcinoma, HepG2 cells, were obtained from Beijing Union Medical Cell Resource Center , and were cultured in minimum essential medium (MEM) supplemented with 10% fetal bovine serum, 1% nonessential amino acids, streptomycin (100 µg/mL) and penicillin (100 U/mL)  C in a 37 °C incubator with 5% CO4 per well. After treatments, CCK8 reagent was added to each well, followed by incubating for another 1 h in a 37 °C incubator with 5% CO2. Then, the absorbance was measured by a microplate reader at 450 nm , and cell viability was expressed as percentage values, as compared with the control group.The HepG2 cells viability was measured by cell counting kit-8 (CCK-8) colorimetric assay , according to the manufacturer’s instructions. Briefly, cells were subcultured in 96-well plates at 1 × 105 cells per well into 6-well plates. After treatments, the cells were washed twice with cold phosphate-buffered saline  and then resuspended in 1× binding buffer at a concentration of 5 × 105 cells/mL. Then, 5 µL annexin V and 10 µL PI were separately added into each tube. After 15 min incubation at room temperature and in the dark, 400 µL of 1× binding buffer was added to each tube. Cell fluorescence was detected by FACS CytoFLEX flow cytometer . Data were analyzed using CytExpert 2.0.0 software .Cells were seeded at a density of 3 × 10The intracellular ROS was analyzed with fluorescent probe 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA), as described previously . After tHepG2 cells were fixed in 4% paraformaldehyde for 15 min, washed with PBS, and incubated with Oil Red O working solution for 15 min at room temperature. Then, lipid droplets were observed and photographed with a microscope . To measure intracellular lipid content by BODIPY lipid probes , HepG2 cells were incubated with 10 μg/mL of BODIPY for 10 min at 37 °C and then stained with DAPI for 3 min. After washing three times with PBS, cells were immediately visualized using a Zeiss LSM-700 confocal microscope .TG contents were determined enzymatically using a commercially available kit according to the manufacturer’s instructions. Intracellular TG content was normalized according to the protein concentration and reported as mmol of TG/g of protein.n =  8) was fed with normal chow  for 17 weeks; HFD group (n =  8) was fed with a high-fat diet  for 17 weeks; HFD +  SFA group (n =  8) received HFD for 17 weeks and were intraperitoneally injected with 10 mg/kg/day SFA from week 14 to week 17 additionally (three times per week). After 17 weeks, mice were fasted overnight and anesthetized. Blood samples were taken from the vena cava. Livers were immediately excised, weighed, and stored at −80 °C for further molecular experiments. The whole experiment was performed in accordance with the Guide for the Care and Use of Laboratory Animals and the research protocol was approved by the Experimental Animal Ethics Committee in China Agricultural University (Permission Number: Aw71211202-4-2).For this study, 6-week-old male C57BL/6J mice were obtained from Beijing Vital River Laboratory Animal Technology Co . Mice were first housed in a controlled environment  under a 12 h light/dark cycle. One week later, mice were randomly allocated to three groups as follows: control group (n = 6) were evaluated under anesthesia using 4% chloral hydrate (4 mL/kg) by MesoMR23-060H-I imaging instrument . The parameters were set as: K space = 192 × 256 mm, time of echo = 13.5 ms, time of waiting = 300 ms, magnet = 0.55 T, section thickness = 3.5 mm, field of view read = 100 mm, field of view phase = 100 mm and number of scans = 8. The body contents can be obtained from the image analysis based on the measurement.Fat distribution and contents of mice .g for 30 min at 4 °C. The supernatants were collected to analyze TG, TC, ALT, and AST contents in the liver using biochemical assay kits according to the manufacturer’s instructions.Liver tissues were homogenized in cold 50 mM Tris-HCl buffer (pH 7.4) containing 1 mM EDTA and centrifuged at 12,000× Mice were fasted for 12 h to analyze the glucose tolerance test after an intraperitoneal injection of 2 g/kg glucose. For the insulin tolerance test, mice were intraperitoneally injected with 1 IU/kg insulin  after 6 h fasting. Blood samples from the tail veil were measured with a glucometer  at the indicated times  after injection.v:v:v) was used for lipids extraction. The samples were vortexed, sonicated 30 s for 3 times, and centrifuged for 10 min at 4000× g. The supernatants were transferred and reextracted as described above. The extracted samples were dried by a gentle nitrogen stream. Then, quantification of ceramides was performed on an Agilent high-performance liquid chromatography (HPLC) system, coupled with a quadrupole-time of flight mass spectrometer (6545 Q-TOF), as described previously [After treatment, cells suspension and liver homogenate were collected separately. After protein determination, 50 µL internal standard solutions were added to the samples, and 2 mL extraction mixture  was conducted to detect hepatocyte apoptosis . The hepWestern blots were performed as described previously  for AMP-p < 0.05) between means were assessed using one-way ANOVA, followed by Duncan’s multiple-comparison test by SPSS 20.0 software .All assays were performed at least in triplicate, and data were presented as means ± standard deviations (SDs). Significant differences  for 24 h, and cell viability was evaluated. As shown in The effect of SFA on PA-induced lipid accumulation was measured by Oil Red O and BODIPY staining, and the extents of staining were spectrophotometrically quantitated. As shown in In order to further understand the possible mechanisms behind the lipid-reducing effect of SFA, the phosphorylated AMPK in HepG2 cells was measured. The level of p-AMPK was reduced to 0.67 after incubation with 300 µM PA for 24 h but returned to 1.17 and 1.22 under co-incubation with 5 or 10 µM of SFA, respectively D,E. The Apoptosis is one of the main types of cell death and plays a fundamental role in the development of multicellular organisms . SaturatExcess oxidative stress is also a well-characterized mechanism of PA-induced hepatic lipotoxicity and the increased intracellular ROS results in hepatocyte apoptosis . Thus, tIn order to explore the effects of SFA on NAFLD in vivo, SFA administration experiments in C57BL/6J mice were conducted. The effects of SFA on body weight and fat distribution were first assessed. After 14 weeks’ treatment with NC or HFD, HFD-fed mice markedly gained body weight, compared with the NC group. Surprisingly, after another 3 weeks of 10 mg/kg SFA intervention, body weight gain was significantly decreased in mice A. This rTo further evaluate the effects of SFA on HFD-induced hepatic lipid deposition, H&E and Oil Red O staining of liver sections were analyzed. The results showed hepatocellular ballooning and hepatocyte fatty degeneration in the HFD group, whereas the lipid droplets were significantly reduced after SFA administration A. Moreovp < 0.05), whereas SFA remarkably recovered these changes. In addition, the increased cleaved caspase 3 was downregulated by SFA (Next, the apoptosis in liver tissues was measured by a TUNEL assay. As shown in d by SFA B,C. Furtd by SFA D. HoweveThis study, for the first time, found that SFA significantly alleviated apoptosis in the development of NAFLD. In addition, a potential new health-related mechanism for SFA was suggested. SFA alleviated lipid accumulation by regulating the AMPK/SREBP1c/FAS signaling pathway. Moreover, antiapoptotic activity, accompanied by a reduction in ceramide production and ROS levels, was evaluated. These results suggest that SFA, as a functional ingredient, could have a substantial impact preventing NAFLD progression.NAFLD patients are shown to have increased lipolysis and carried FFAs to the liver. Excessive FFAs accumulation is the principal contributor to lipotoxicity because of the potential cellular toxins and leads to excessive lipid steatosis . TherefoNAFLD is mainly characterized by fat deposition in hepatocytes, visible at light microscopy as small droplets inside the cytoplasm. Thus, the therapy based on reducing lipid accumulation is ideal for treating NAFLD . It is rApoptosis is defined as programmed cell death and is accompanied by many specific morphological features, such as cell shrinkage, nuclear fragmentation, and chromatin condensation, which is another important mechanism in the progression of NAFLD. Hepatocyte apoptosis is often observed in NAFLD studies ,39. WhenIn conclusion, the data reveal that SFA suppresses PA-induced lipid accumulation and apoptosis in HepG2 cells, as well as HFD-induced liver injury and steatosis in C57BL/6J mice. This work contributes a new understanding with regard to the molecular mechanisms underlying the inhibitory effect of SFA on hepatic lipid deposition and apoptosis. This study suggests that SFA may be a potential candidate for the amelioration and prevention of NAFLD.


Antimicrobial resistance (AMR) is a global public health concern because of its fast spread. India, one of the world’s top consumer of antibiotics and second most populated country has its unique constraints of social, cultural and economic strata. The continual self-medication, use of antibiotics for the growth promotion in animals, and accumulation of residual antibiotics in the environment challenge the implementation of AMR containment policy. Hence, the present review attempts to delineate the influence of antibiotics abuse on the human, animal and environmental health under the realm of one health. It was based on the literature search using public databases to highlight the rapid surge in the burden of AMR in India affecting various sectors and/or ecosystems in India. It was found that the irrational and overuse of antibiotics in different sectors have led to the emergence of extended antimicrobial resistance wherein the environment acts as a reservoir of antibiotic resistance genes (ARGs); completing the cycle of contamination and recontamination. There are efforts by government policy makers to reduce the burden of AMR in the country to reduce the health risks, through the One Health approach. Parallel efforts in educating healthcare professionals, strict legislation for pharmacies and pharmaceutical companies should be prioritize. At the same time surveillance of newly emerged AMR pathogens, prioritising research focusing on AMR, and awareness camps or programs among the local population is critical while addressing the consequences of spared of AMR in India. Discovery of the antimicrobial agents in the early twentieth century was a breakthrough to safeguard the public health from the microbial infection. Propensity of these compounds to kill pathogens influenced the discovery of novel antimicrobials. However, since the 1970s, soon after the discovery of the fluoroquinolones, no major antibiotics have been introduced , 3. The Bacteria are ubiquitous in nature; thriving in soil, water and air and, interconnectedness of ecosystems, featuring human, animals and environment collectively indicates that the burden of AMR is multifaceted in nature . AntibioblaNDM-1  and associated controversy necessitated the policymakers to initiate the development of AMR containment-related policies for India in 2011 . Mo. MoAcineectively . Thus, rc health , 29.Fig.E. coli or Salmonella species. Furthermore, there have been molecular investigations suggesting extended-spectrum beta-lactamase (ESBL)-producing and colistin-resistant E. coli from the poultry [The ever-increasing demand of animal-based food products compelled the producers to increase the usage of antibiotics and growth-promoting factors in the animal feed. The higher usage of antibiotics aids the better health of animals and also enable industries to abandon the responsibilities of maintaining hygiene and sanitation. Such ill-usage of antibiotics in the animal feed leads to bioaccumulation which was supported by observed traces of antibiotics in animal-based food products . Majorit poultry . Moreove poultry . The ant poultry . MoreoveStaphylococcus aureus have been isolated from the animals suffering from mastitis [Vibrio species and members of family Enterobacteriaceae [MDR strains like Vancomycin-resistant mastitis . Furthermastitis , 36. Mormastitis . Similarmastitis . Excessimastitis , 17. Stueriaceae , 17. Preeriaceae . Additioeriaceae –17, 33.The finding of antimicrobial-resistant microorganisms in humans, poultry, cattle and fishes reveal possible transmission and sharing of such microorganisms and antibiotic resistance genes (ARGs) between these organisms. However, the underlying mechanism of transmission and establishment of AMR is not well understood . Thus, ablaNDM-1, blaOXA-48, blaCTX-M and mcr-1 genes [NDM-1 genes are responsible for the development of drug resistance in pathogens and also provide an advantage to evolve as a superbug [Sewage plants receiving untreated or inadequately treated hospital and industrial effluents are a potential reservoir of AMR and ARGs. Discharge of untreated sewage into the rivers is a another major factor contaminating the river and facilitating the spread of AMR. Recent studies from India have indicated that the hospital waste carries significantly higher proportion of antibiotic residues, particularly the traces of broad-spectrum antibiotics . In the -1 genes –43. blaNsuperbug . Similarsuperbug , 41. Thesuperbug .blaNDM-1 were monitored during the seasonal pilgrimage on the bank of Ganga. The authors reported a 20-fold increase in prevalence during the pilgrimage [Acinetobacter, Corynebacterium and Brevibacterium which was isolated during the event show higher antimicrobial resistance [Corynebacterium godavarianum, a novel bacterium isolated during the Kumbh Mela 2015, was found to confer resistance against an array of antibiotics supporting the observed increase in antimicrobial resistance during the mass bathing event [India is a country of festivals involving several cultural events during the year, one such event is the Kumbh Mela. It is considered as the world’s largest mass gathering event attracting millions of pilgrims for the duration of three months , 46. Thelgrimage . Similarlgrimage , 50. Antng event .Neisseria meningitidis serogroup W135 in 1968, 1987, 2000 and 2001, H1N1 pandemic in 2009, Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in 2012, etc. Additionally, such a large participation also elevates the risk of stampedes or crush injuries, hence necessitates intensive planning and preparedness of the event to minimize the risk to the public health [India is one of the ~ 184 countries that take part in Hajj pilgrimage at the Mecca, Saudi Arabia. Participation of pilgrims from these diverse countries has been associated with the morbidity and mortality at the event. In past, the Hajj has witnessed the burden of communicable diseases like, c health –54. Massc health , 52, 55.c health , 52. ThuThe striking observations of threatened humans, animal husbandry and fishery and, environment health due to growing antimicrobial resistance raise a serious concern Fig. . Self-meTo address the ever-growing challenge of antimicrobial resistance and to safeguard public health, officials keep on upgrading the local policies by following the strategies outlined by WHO. The controversies related to the NDM-1 drew the attention of policy makers to initiate the National Policy on Containment of AMR in 2011 . The MinSimilarly, to safeguard the animal health, Food Safety and Standards Authority of India (FSSAI) has developed norms for the rational use of antibiotics. The FSSAI has banned the use of several antibiotics that could harm the fisheries and those used in the seafood processing . FurtherThe irrational and overuse of antibiotics in different sectors of India i.e., humans, food, animals and environment are responsible for the ever-increasing antimicrobial resistance (AMR), however, it has received little attention compared to its drastic impacts. Mitigation of the growing antimicrobial resistance would require strategies focusing on the one health approach. Initiative of the Indian government such as NAP-AMR policy along with the continued surveillance, awareness camps, and cross-continental research and training would enable combating the issue of AMR. Initiatives like designated AMR repositories by the Department of Biotechnology, Government of India at the National Centre for Cell Science, India would be helpful to generate and validate the emergence of AMR-related data in different sectors of India.


D) and the crystal growth rate (G) of the crystalline component. There are both inter-lamellar and inter-fibrillar structures when PVDF blended with lower-molecular-weight PMMA. With increasing molecular weight of PMMA, the decrease in crystal growth rate (G) dominates the enhanced exclusion behaviors of PMMA, resulting in bigger pores after extraction. Our results are significant not only for the basic understanding of crystallization in polymer blends, but also for the fabrication and structure control of porous structures based on crystallization templates.In miscible crystalline/amorphous polymer blends, the exclusion behaviors of the latter with various molecular weights during the crystallization of the former were investigated by the combination of SAXS and DSC by taking a PVDF/PMMA blend as an example. The ratio between internal crystallinity from SAXS and overall crystallinity of the entire blend from DSC was employed to characterize the exclusion of PMMA. Our results indicate that the molecular weight of the amorphous component produces a remarkable influence on the diffusion coefficient ( Conversely, the diluent will be excluded to the regions among lamellae stacks or spherulites when the ratio is high. According to this parameter, Yan et al. investigated the composition and isothermal temperature dependences of the exclusion behaviors in poly (butylene succinate)/poly (butylene adipate) (PBS/PBA) blend [Miscible crystalline/amorphous polymer blends have attracted much attention because of their wide applications ,3,4,5,6.r blends . In the A) blend . The res2 groups of PVDF [In the investigation of the localization of amorphous components, considerable advances have been made by taking poly (vinylidene fluoride)/poly (methyl methacrylate) (PVDF/PMMA) as an example. PVDF/PMMA is a typical miscible crystalline/amorphous blend in the case of atactic- and syndiotactic-PMMA. The miscibility can be attributed to the favorable interactions between the carbonyl group of PMMA and the CH of PVDF ,14. The  of PVDF ,16,17. T of PVDF .X1) from SAXS is defined as the ratio between d/L, where d and L are lamellae thickness and the long period of the crystal, respectively; the overall crystallinity of the entire blend  from DSC can be calculated from the enthalpy values. The schematic representation of X1 and X2 is shown in X1) calculated based on the lamellae thickness and long period does not contain the excluded part of PMMA (red part in X2) considers the amorphous PMMA. However, it is impossible to evaluate the exclusion behaviors of PMMA based only on this parameter since the localization of PMMA is not clear. Hence, the ratio between X1 and X2 can act as an effective measure for the extent of PMMA exclusion. A higher value of X1/X2 corresponds to the enhanced exclusion behavior of PMMA. Using this parameter, Lin et al. clarified the role of molecular weight of crystalline component in determining the segregation morphologies [G) and diffusion coefficient (D) [Less attention has been paid to the inter-lamellar regime since its size is too small to track using microscopes. As a result, small-angle X-ray scattering (SAXS) has been introduced for the investigation of exclusion behaviors in lamellar scale ,20. To ahologies . The molient (D) ,24,25. Tient (D) ,20,24, wMw = 209,000 g/mol, PDI = 2.0, ρ = 1820 kg/m3) was supplied by Kureha Chemicals . Four kinds of PMMA (CAS: 9011-14-7) with different molecular weights were purchased from Sigma-Aldrich  and named PMMA-1, PMMA-2, PMMA-3, and PMMA-4  were provided by Lingfeng Chemical Reagent Co., Ltd. .PVDF . All samples were cut into cuboid shapes (10 × 6.30 × 1 mm) and heated from −50 to 200 °C with a heating rate of 3 °C/min. A tensile mode with an amplitude of 10 μm and a frequency of 5 Hz was applied.The thermodynamic and crystallization behaviors of PVDF/PMMA blends were investigated by a DSC Q2000  under nitrogen flow. Specimens were heated from 30 to 200 °C with a rate of 10 °C/min and held at 200 °C for 5 min to eliminate thermal history, and then cooled to 30 °C with a cooling rate of 3 °C/min. Before tests, the temperature and heat flow were calibrated with sapphire and pure indium, respectively. The glass transition temperature of PMMA was also evaluated by means of a DSC.The crystallization dynamics of the PVDF/PMMA blends were investigated with the help of POM measurement . The POM was fitted with a charge coupling device (CCD) camera interfaced with a computer. The observed specimens were prepared by droplet drying from polymer blend solutions on glass sheets. A Linkam LTS 350  hot stage was used to control the temperature. The samples were melted at 200 °C for 5 min to erase thermal history and then quickly cooled to 150 °C for isothermal crystallization of PVDF.Lamellar thickness, long spacing, thickness of the amorphous region, and crystallinity for the blends were calculated based on the information collected from a beamline BL16B1 (Shanghai Synchrotron Radiation Facility). The wavelength of the X-ray was 1.24 Å. The sample-to-detector distance was 1900 mm.The removal of PMMA in blends was verified by thermogravimetric analysis . The temperature was raised from 30 to 650 °C at the rate of 10 °C/min in a nitrogen atmosphere.The samples were fractured in liquid nitrogen, and the fracture cross-section coated with gold was examined by field-emission scanning electron microscopy  at an accelerating voltage of 5 kV. Pore size statistics were carried out by a Nano Measurer . Relevant data were obtained through the pore size statistics and analysis of porous PVDF phases in SEM images. The porous PVDF samples were prepared from the PVDF/PMMA blends after extraction with chloroform in a Soxhlet extraction device  for 72 h.Tg) of the polymer blend observed by DMA measurement was employed to evaluate the miscibility of two components. The dynamic loss for pure PVDF and PVDF/PMMA blends with different molecular weights of PMMA is shown in Tg was observed for pure PVDF, located at approximately −31 °C . The Tg of the blend increases with the increase in the molecular weight of PMMA [Tm depression effect from Nishi and Wang [The miscibility between PVDF and PMMA with different molecular weights is the basis of discussing the effect on its exclusion behaviors. Here, the glass transition temperature  of PVDF spherulites diameter in blends with different molecular weights can be calculated based on the POM images. In the same way, it was easy to obtain magnitudes of G from all specimens, which are shown in G value in binary blends gradually decreased from 1.51 ± 0.18 to 0.10 ± 0.01 μm/min upon increasing molecular weight of PMMA, which can be attributed to the higher viscosity caused by the increase in PMMA molecular weight. Furthermore, the growth rate of PVDF spherulites is in agreement with the crystallization kinetics of PVDF in their blends with PMMA shown in The evolution of PVDF spherulites at 150 °C were tracked by means of POM (0 min was the starting point for the observation of spherulites). The spherulite growth of PVDF in PVDF/PMMA binary blends is shown in 3, respectively. The crystalline phase density (ρc) and amorphous phase density (ρa) of PVDF are 2.00 and 1.74 g/cm3, respectively. According to the calculation, the PVDF/PMMA blend system meets the requirements discussed above [K(z) were calculated by the Fourier transformation of scattering curve according to Equation (1) [I and q are scattering intensity and characteristic wave number, respectively. The Lorentz-corrected SAXS curves of samples are shown in q value with increasing Mw of PMMA in the PVDF/PMMA blends. This means that the long period of PVDF decreases because of the enhanced exclusion of PMMA chains. The obtained correlation function K(z) is shown in d), the average long spacing (L), and the average thickness of the amorphous region (AM) for the blends and the internal crystallinity (X1 = d/L), which are listed in d and AM decrease with increasing molecular weight of PMMA, resulting in a smaller long period. As for the internal crystallinities (X1) calculated based on the average lamellar thickness d and the average long spacing, the dramatic in of amorphous thickness accounts for the bigger internal crystallinities (X1) of binary blends with increasing PMMA molecular weight.The necessary condition for analyzing the interlayer inclusion and interlayer exclusion structure of binary blends by SAXS is that the density difference between the two components is greater than the density difference between the crystalline and amorphous phases of each component. The densities of PVDF and PMMA are 1.78 and 1.19 g/cmed above . In ordetion (1) (1)Kz=[∫Tm-depression effect [d and L in wX) of the entire blend can be calculated by the enthalpy values:wX, from DSC) into volume crystallinity (X2). The relationship between them in the blends of PVDF and PMMA is as follows:X2) roughly decreases. The ratio between X1 and X2 can act as a good parameter to describe the exclusion behaviors of amorphous PMMA. The molecular weight dependences of exclusion behaviors are shown in The heating DSC curves of PVDF/PMMA blends with different molecular weights that were fully crystallized at 150 °C are shown in n effect ,35. FurtX1 and X2 should exhibit close values. On the contrary, X1 shows a higher magnitude relative to X2 when all PMMA chains are excluded from inter-fibrillar regions. In other words, lower (or higher) magnitudes of X1/X2 correspond to more (or less) PMMA locating in inter-lamellar regions. It is obvious that the molecular weight of PMMA produces remarkable effects on the ratio  introduced by Keith and Padden was employed in this work. D and G are the diffusion coefficients of the amorphous component and the crystal growth rate of the crystalline component, respectively. With increasing molecular weight of PMMA, both the growth rate (G) of PVDF (D) of PMMA decrease due to the improved entanglement effect and higher viscosity. The competition between G and D determines the δ and exclusion of amorphous component. In this work, the decrease in G dominated the parameter of δ and the resultant exclusion behaviors of PMMA, resulting in a higher fraction of inter-fibrillar structures in the case of higher molecular weight. That is to say, if the growth rate of lamellae decreases, the chain of PMMA still has sufficient time to escape from the inter-lamellar region even when the diffusion coefficient of PMMA becomes lower because of the higher molecular weight.If all amorphous PMMA chains locate among PVDF lamellae, he ratio  and the  of PVDF  and diffIn our previous work, crystalline/amorphous bi-continuous structures in their miscible blends fabricated based on crystallization template have been validated by selective solvent extraction ,37,38,39X1  and X2 , it is obvious that the molecular weight of PMMA produces a remarkable effect on its exclusion behaviors via varying growth rate (G) and diffusion coefficient (D). As shown in G). In this process, there is enough time for PMMA chains to diffuse and escape from the crystal growth front. As a result, PMMA enriches in inter-fibrillar regions, which is the reason for the bigger pores in PVDF upon chloroform etching.According to the crystallinity ratio of G dominates the parameter of δ and the resultant exclusion behaviors of PMMA, resulting in a higher fraction of inter-fibrillar structures in the case of higher molecular weight. The localization of PMMA was validated by the porous structures after the extraction of PMMA by chloroform. With increasing molecular weight of PMMA, more PMMA is excluded from the inter-lamellar region, resulting in bigger pores. Our results are significant not only for the basic understanding of crystallization in polymer blends but also for the fabrication and structure control of porous structures based on crystal templates.In this work, PVDF/PMMA, a typical miscible crystalline/amorphous blend, was employed to investigate the effect of molecular weight of PMMA on its exclusion behaviors during the isothermal crystallization of PVDF. Our results indicate that the molecular weight of PMMA produces remarkable effects on both the growth rate of PVDF and the diffusion coefficient of PMMA. In these two parameters, a decrease in


During the COVID-19 pandemic, the UK government implemented a series of guidelines, rules, and restrictions to change citizens’ behaviour to tackle the spread of the virus, such as the promotion of face masks and the imposition of lockdown stay-at-home orders. The success of such measures requires active co-operation on the part of citizens, but compliance was not complete. Detailed research is required on the factors that aided or hindered compliance with these measures.To understand the facilitators and barriers to compliance with COVID-19 guidelines, we used structural topic modelling, a text mining technique, to extract themes from over 26,000 free-text survey responses from 17,500 UK adults, collected between 17 November and 23 December 2020.The main factors facilitating compliance were desires to reduce risk to oneself and one’s family and friends and to, a lesser extent, the general public. Also of importance were a desire to return to normality, the availability of activities and technological means to contact family and friends, and the ability to work from home. Identified barriers were difficulties maintaining social distancing in public , the need to provide or receive support from family and friends, social isolation, missing loved ones, and mental health impacts, perceiving the risks as low, social pressure to not comply, and difficulties understanding and keep abreast of changing rules. Several of the barriers and facilitators raised were related to participant characteristics. Notably, women were more likely to discuss needing to provide or receive mental health support from friends and family.The results demonstrated an array of factors contributed to compliance with guidelines. Of particular policy importance, the results suggest that government communication that emphasizes the potential risks of the virus and provides simple, consistent guidance on how to reduce the spread of the virus would improve compliance with preventive behaviours as COVID-19 continues and for future pandemics.The online version contains supplementary material available at 10.1186/s12889-021-12372-6. To tackle the COVID-19 pandemic, governments focused on reducing transmission of the virus by influencing citizens’ behaviour. Governments mandated the wearing of face masks in public places, recommended social distancing, promoted regular handwashing, and even ordered the closing of businesses, prohibited household mixing, and implemented stay-at-home “lockdown” orders. These measures (where followed) are effective at reducing infection rates , 3, and Policymakers’ beliefs about compliance behaviour – the extent to which citizens comply, for how long, and in which contexts – influenced the measures that have been put in place. For instance, the possibility of “risk compensation”  – indiviGiven the importance of citizens’ behaviour for tackling COVID-19 – and previous – pandemics, a large literature has grown on the determinants of compliance, including on the motivations, barriers, and facilitators of compliance and the personal and situational characteristics associated with high compliance levels . RecQualitative studies offer an opportunity to deepen our understanding of compliance behaviour, allowing for greater flexibility in the exploration and identification of relevant phenomena. Recent qualitative studies from the UK found evidence of “alert fatigue”, with individuals expressing difficulty keeping abreast of frequently changing guidelines and, as a result, inadvertently breaking (or knowingly bending) government rules , 26. It Text-mining approaches offer a middle ground, allowing for the extraction of themes from large-scale free-text data that can then be related to document metadata, such as the date the text was written and the characteristics of its author . Participants can provide spontaneous information, which can be summarised quantitatively and analysed as any other quantitative variable . In this study, we used structural topic modelling (STM)  – a texthttps://github.com/UCL-BSH/CSSUserGuide. The study was approved by the UCL Research Ethics Committee  and all participants gave informed consent.We used data from the COVID-19 Social Study; a large panel study of the psychological and social experiences of over 70,000 adults (aged 18+) in the UK during the COVID-19 pandemic. The study commenced on 21 March 2020 and involved online weekly data collection for 22 weeks with monthly data collection thereafter. Data collection is still ongoing. The study is not random and therefore is not representative of the UK population, but it does contain a heterogeneous sample. The sample was recruited using three primary approaches. First, convenience sampling was used, including promoting the study through existing networks and mailing lists , print and digital media coverage, and social media. Second, more targeted recruitment was undertaken focusing on (i) individuals from a low-income background, (ii) individuals with no or few educational qualifications, and (iii) individuals who were unemployed. Third, the study was promoted via partnerships with third sector organisations to vulnerable groups, including adults with pre-existing mental health conditions, older adults, carers, and people experiencing domestic violence or abuse. Full details on sampling, recruitment, data collection, data cleaning and sample demographics are available at Since the pandemic started, what has been encouraging or helping you to follow the guidelines, even if only to a partial extent?If you have not been entirely following the guidelines, what are the factors that have been hindering you or acting as obstacles?A one-off module on compliance behaviour was included in the survey between 17 November and 23 December 2020. The module included two free-text questions on facilitators and barriers to compliance, respectively:We conducted analyses for both questions, separately. Twenty-five thousand fifty-one individuals participated in the data collection containing the free-text survey module (34% of participants with data collection by 23 December 2020). Responses to the free-text questions were optional. Eighteen thousand seven hundred forty-two participants provided a response to the question on facilitators of compliance (74.82% of eligible participants) and 11,902 participants recorded a response to the question on barriers (47.51% of eligible participants). Of these, 16,512 (88.1%) and 9720 (81.6%) participants provided a valid record, the definition of which is provided below.The period 17 November – 23 December 2020 overlapped with the beginning of the second wave in the UK and the announcement, and start of the rollout, of a vaccine against COVID-19. Government rules changed across the study period. A description of the changes in these rules is provided in the We performed topic modelling using unigrams (single words). Responses were cleaned using an iterative process. We excluded responses containing fewer than five words and removed words that appeared in fewer than five responses. We also removed common “stop” words  from the analysis. We used complete case data so excluded a small number of participants with missing data on any covariate used in the analysis. Spelling mistakes were identified with the hunspell algorithm , amendedWe performed several quantitative analyses. First, as not all participants chose to provide a response, we ran a logistic regression model to explore the predictors of providing a free-text response. Second, we used STM, implemented with the stm R package , to extrWe ran STM models from 2 to 30 topics for each question and selected the final models based on visual inspection of the semantic coherence and exclusivity of the topics and close reading of exemplar documents representative of each topic. Semantic coherence measures the degree to which high probability words within a topic co-occur, while exclusivity measures the extent to that a topic’s high probability words have low probability for other topics. After selecting a final model, we carried out two further analyses. First, we decided upon narrative descriptions for the topics based on high probability words, high “FREX” words (a weighted measure of word frequency and exclusivity), and exemplar texts (responses with a higher proportion of text estimated for a given topic). Second, we ran regression models estimating whether topic proportions were related to author characteristics defined above. Third, we ran a regression model estimating whether self-reported adherence to COVID-19 guidelines  was related to topic proportions, to explore which barriers to compliance may make compliance particularly challenging.https://osf.io/nf4m9/.Data cleaning and analysis was carried out by LW. LW and EP selected the number of model topics and LW, EP, AS and DF agreed upon narrative titles for the topics. The datasets generated and/or analysed during the current study are not publicly available due stipulations set out by the ethics committee but are available from the corresponding author on reasonable request. The code used in the analysis is available at The funders had no final role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication. All researchers listed as authors are independent from the funders and all final decisions about the research were taken by the investigators and were unrestricted.Sixteen thousand five hundred twelve participants provided a valid free-text response to the question on facilitators, and 9720 individuals provided a valid free-text response to the question on barriers . The median response length for the valid free-text responses was 18  for the question on facilitators and 22  for the question on barriers. Descriptive statistics for respondents are displayed in Table SA word cloud of the twenty most frequently used words for each question is displayed in Fig. A 14-topic solution was chosen to categorise facilitators of complying with guidelines. Exemplar quotes, topic descriptions, and topic proportions are presented in Table Several of the topics related to desires to protect oneself and others from COVID-19. The largest topic, Topic F1 , included text on worries about catching the virus and passing it on to family members, particularly elderly parents. Topics F2  and Topic F6  related to protecting high risk and clinically vulnerable individuals, specifically. Exemplar texts typically referred to reducing risk for oneself and for one’s family and to a lesser extent the wider population. Similar to Topic F1, Topic F8  related to protecting family and friends (topics differ in specific words used). Topic F13  related to reducing the burden on the National Health Service and its workers.Prosocial motivations were also identified in Topic F5 , which explicitly couched compliance as a matter of social responsibility, civic duty, or simply a matter of “common sense”. Topic F4  identified individuals predisposed to follow rules in general, though this topic also surfaced responses from individuals describing others’ rule following as a motivator. Topic F9  related to desires to hasten the end of the pandemic and return to life as normal.A role of media coverage and scientific information was identified in Topic F3 , which contained text on news and media reports, including briefings from Government ministers and scientists. Topics F7, F10 and F11 each related to factors that made compliance easier. Topic F7  included positive statements on the availability of hand sanitiser in shops and on reminders to wash hands and wear masks in public places. Topic F10  included responses from individuals who had been able to work from home (or who had lost work and so did not need to travel to a workplace) and also on the availability of support bubbles, allowing participants to receive or give support to family and friends. Topic F11  related to activities that had improved quality of life during lockdown, including walking in nature and participating in online activities, such as arts and talking to family members over Zoom.The final two topics, Topics F12 and F14, contained irrelevant material or identified texts on a heterogeneous set of themes. Topic F12  included discussion of personal symptoms from COVID-19 or experiences with the test and trace system. Responses appeared to arise from participants expanding on their response to preceding questionnaire items. Topic F14  identified responses generally covering themes from the other topics, typically using slightly different phrasing.Figures There were a number of differences according to age Fig. . YoungerThere were also several differences according to personality traits Fig. . IndividFinally, there were several differences according to participants’ further demographics as well as their socio-economic characteristics, health and confidence in government Fig. . Female We selected a 14-topic solution for the responses on barriers to compliance. Short descriptions are displayed in Table Several topics related to difficulty understanding current rules. Topic B11  identified individuals who had difficulty keeping abreast of (frequently changing) rules or who could not understand the logic behind the rules . Some responses identified in this topic also questioned the lack of flexibility in rules or the allowance for individuals to apply common sense. Topic B13  related to challenges arising from the variation in rules around the UK, particularly for those living near borders, such as that between England and Wales. One frequently noted issue was accessing essential services located on the other side of a border. Topic B14  included responses that described general confusion about what the rules were, though this topic also identified individuals who stated following guidelines to the best of their ability.  Criticism of the government was voiced in Topic B7 , with individuals noting a lack of confidence or trust in the government’s decisions or motives, including in Boris Johnson’s leadership. Several participants also expressed anger at Dominic Cummings – a senior Government advisor – and the decision to keep him in post following reports in May 2020 that he had broken lockdown rules.Several topics related to the negative impact of compliance on the lives of participants and their family and friends. Topic B3  related directly to the impact of restrictions on mental health for the participant themselves and their family and friends. The topic also identified responses from individuals who had extended support bubbles beyond the rules to provide or receive support from loved ones. This included both emotional and practical support . Topic B6  related to difficulties due to missing family and friends, particularly when the weather turned poor and meeting outside became a challenge. Topic B12  identified responses from individuals who lived alone and had struggled due to the resulting social isolation. Some individuals described breaking lockdown rules on occasion for human contact. Topic B4  also identified individuals who had occasionally broken lockdown rules to improve wellbeing, but also identified individuals breaking rules to provide support to family members  or out of temporary necessity .Practical barriers to compliance were reflected in Topic B2  which related to difficulties with face masks or using hand sanitiser. Several respondents stated forgetting to wear masks or use sanitiser or noted discomfort from the use of these due to existing health conditions. Finally, Topic B9  identified individuals who did not comply with guidelines due to perceptions that risks were low, for instance, due to previous infection with COVID, low caseloads in the local area, or beliefs that government statistics were exaggerated. The topic also identified a number of responses of individuals who had not gotten tested when displaying symptoms, either due to lack of availability or beliefs that PCR tests were not accurate.Figures There were also a number of differences according to personality traits Fig. . NotablyFinally, there were several differences according to participants’ demographic and socio-economic characteristics, health and confidence in government Fig. . Female The results of regressions estimating the average level of self-reported compliance according to topic proportions are displayed in Fig. Facilitator topics related to desires to protect the vulnerable (Topic F6) or high risk (Topic F2) or reduce the likelihood of catching and transmitting COVID-19 were associated with highest compliance levels. Following the rules (Topic F4), desire to protect the NHS (Topic F13) and acting out of social responsibility (Topic F5) were related to lowest average compliance levels. For barriers to compliance, perceiving COVID-19 as representing a low risk (Topic B9), living alone (Topic B12), lack of trust in government (Topic B7) and finding the rules to be too complex (Topic B11) was related to lowest compliance levels. Following the guidelines completely (Topic B5), confusion around the rules (Topic B14) and facing special circumstances (Topic B4) were related to higher than average compliance.Using free-text data from over 17,500 adults, we identified several facilitators and barriers to compliance with COVID-19 guidelines, 8 months after lockdown measures were implemented in the UK. For facilitators of compliance, a sizeable proportion of text was related to desire to reduce risks for oneself, one’s family and friends, and – to a lesser extent – the general public and the NHS and its workers, specifically. Some participants also spoke of being motivated by a sense of responsibility, a desire to return to life as normal, or acting from a predisposition to follow rules in general. For barriers to compliance, a substantial proportion of text was related to other people making compliance difficult, either by getting too close in public or workplaces, putting social pressure on participants to violate guidelines, or acting as a demotivator when their non-compliance was observed by participants. Participants also spoke of the emotional toll of complying with guidelines, particularly among those who lived alone, missing family and friends, and of the necessity of providing support to loved ones. Interestingly, participants also spoke of breaking rules on occasion, for instance when social isolation had gotten too great. A large portion of text was also devoted to issues with the guidelines themselves. Participants found guidelines confusing and often did not see their logical basis. The variation in rules across time and geographic areas was a particular issue. Some participants also discussed a lack of trust in government and expressed anger at the decision to keep Dominic Cummings in his position as government advisor after he broke lockdown rules. Last, some participants discussed believing COVID-19 to be low risk as a reason for not complying. Individuals who spoke about this had the lowest self-reported compliance overall.Several theoretical frameworks have been used to understand individual differences in compliance with preventive behaviours. One fruitful framework has been the COM-B model , 35. TheIn Table The topics that participants discussed were related to participant characteristics. Notably, younger individuals were more likely to mention social pressures as a barrier to compliance, and older individuals were more likely to discuss confusion around rules. Extraverted individuals and women were more likely to report emotional challenges and lack of social contact as a barrier to compliance, while conscientious individuals were more likely to state complying with guidelines completely. The topics discussed were also likely to have differed by the date the free-text data were collected, though this was not directly tested in the present study. In particular, individuals may have been less likely to discuss protecting the vulnerable when COVID-19 cases were low in early November, and may have expressed more difficulty with isolation as Christmas approached following Government announcements that household mixing would be limited. The date on which data were collected is also likely to have influenced associations with our measure of self-reported compliance, given that the latter item regarded present compliance. In other work, we have shown self-reported full compliance tracked caseloads whilst become slightly less common over time .Our results are consistent with those of Coroiu et al. , who stuOur finding that a number of participants violated guidelines on occasion for emotional reasons is important in light of debates on behavioural fatigue , 10, 12.Our finding that many participants had struggled to understand or keep abreast of changing rules  was consistent with previous qualitative work showing difficulties understanding government messaging , 26, 38.Also of policy interest was the role of mental health and the need to receive or provide support to family and friends as a barrier to compliance. Social bubbles were not introduced immediately by the governments of UK. The results here suggest this rule may have caused stress or been ignored by some households. Further, some participants complained that support bubbles were of insufficient size when introduced. More flexibility may have improved the wellbeing of individuals requiring support, though an issue is that flexibility could increase the complexity of rules, which as discussed, can raise its own issues for facilitating high compliance.This study had several strengths. We used rich qualitative data from over 17,500 UK adults representing a wide range of demographic groups. Using structural topic modelling, we were able to combine qualitative and quantitative approaches, to identify unique facilitators and barriers to compliance with guidelines in the UK, and to assess how response topics differed according to participant characteristics. Our results have several policy implications and showed (to our knowledge) unique evidence of occasional, isolated rule violations among some of the general public. The results also add detail to previous quantitative results showing a link between age, personality traits and compliance behaviour , 21. TheNevertheless, this study had several limitations. Not all of the topics identified a single theme consistently. Associations with participant characteristics could be driven or biased by idiosyncratic texts. We used a convenience sample that, though heterogeneous, was unrepresentative of the UK population and, further, respondents to the free-text question were biased towards the highly educated. Response biases could also have generated bias in the topic regression results. The sample was drawn from an ongoing study with frequent follow-ups (weekly from March to August 2020 then monthly thereafter). Participation in a study focused on COVID-19 could feasibly have influenced individuals’ compliance behaviour. As we focused on topics offered spontaneously by respondents, a participant not writing about a particular topic does not necessarily imply that the participant does not agree with its sentiment, though our assumptions is that the proportion of text devoted to a topic is related to its perceived importance. Finally, while we included a wide set of predictors in our structural topic models, many relevant factors were unobserved and several were likely to be measured with error or insufficient granularity. For instance, we measured ethnicity as white or non-white and geographic variation at the country level, while there is more heterogeneity within these groups. Associations may therefore be biased by unobserved confounding.We identified several facilitators and barriers to compliance. Of particular importance for facilitating compliance was concerns about the risk of COVID-19 for oneself and one’s family and friends, while important barriers to compliance were problems maintaining social distance in public spaces and difficulties understanding and keeping abreast of government rules. The results suggest that government communication that emphasises the potential risks of COVID-19 and provides simple, consistent guidance on how to reduce the spread of the virus would improve compliance with preventive behaviours. Investments in managing or reorganising public space – particularly in supermarkets – so that social distancing is encouraged could also have significant positive effects. While a large literature has related individual characteristics to preventive behaviour, our results give fresh insight into the wider contextual issues that are important for compliance.Additional file 1.