In the current study, we used RNAi-constructed CmTFL1 (white-flowered) and CmSVP overexpressed (yellow flowered) transgenic plants of previously conducted two studies for our experiment. Cross hybridization was performed between these intergeneric transgenic and non-transgenic plants of the winter-growing chrysanthemum selection “37” (light pink flowered). The transgene CmSVP was confirmed in F1 hybrids by RT-PCR analysis, whereas hybrids of CmTFL1 parental plants were non-transgenic. Besides this, quantitative real-time PCR (qPCR) was used to explain the molecular mechanism of flower development using reference genes. Intergeneric and interspecific hybrids produced different colored flowers unlike their respective parents. These results suggest that generic traits of CmSVP overexpressed plants can be transferred into F1 generations when crossed with mutant plants. This study will aid in understanding the breeding phenomenon among intergeneric hybrids of chrysanthemum plants at an in vivo level, and such transgenics will also be more suitable for sustainable flower yield under a low-light production system.Gene silencing is the epigenetic regulation of any gene in order to prevent gene expression at the transcription or translation levels. Among various gene silencing techniques, RNA silencing (RNAi) is notable gene regulation technique that involves sequence-specific targeting and RNA degradation. However, the effectiveness of transgene-induced RNAi in F Chrysanthemum ×grandiflorum Tzvelve.) is a well-known flower originating from China and other Asian countries . Chrysanthemum is the second most important floriculture crop in the worldwide floriculture trade [Chrysanthemum  is one of the perennials belonging to the temperate climatic zone with an optimum temperature range between 18–21° for growth and development [Chrysanthemum  Kitamura (2n = 2x = 18) and C. indicum var. procumbense (Lour.) Kitamura (2n = 4x = 36) resulted from florist chrysanthemum (2n = 6x = 54) [n = 54 ± 7∼10), even within a genotype; formation of fragmented chromosomes during mitosis; substantial genome size (25 pg DNA/cell); sporophytic self-incompatibility barrier, inbreeding depression, and genetic load [Chrysanthemum species are originated in Japan [The origin of novel potted and ornamental chrysanthemum plants is from interspecific crosses between wild natives of China, which is considered as the epicenter of the genus anthemum . Crossin6x = 54) . Neverthtic load ,18 althotic load ,20,21,22in Japan .1 hybrids of chrysanthemums is rare [CmTFL1 transgenic Chrysanthemum morifolium var ‘Fenditan’ and CmSVP transgenic overexpressed plants of ‘Ganjue’ cultivar taken from our previously conducted study [2 = 0.82) and modified physical parameters along with flower color were demonstrated.The continuous development and innovation of plant molecular biology provide a new way to improve the characteristics of ornamental plants. Novel genetic, biological, and molecular techniques are required to fulfill consumer demands. For this purpose, interspecific hybridization could be a great source in which the plants that belong to two unlike non-compatible species are crossed. Useful or favorable traits are transferred from wild relatives to cultivated ones or between two different species. Interspecific hybridization has been widely used in the Brassicaceae family to transfer favorable genetic traits to a wide germplasm ,25. Many is rare ,28,29. Ied study  with one1 hybrids, followed by a gradual increase at 40% light strength. Interestingly, transgenic hybrids of the cross 37 × G and G × R were significantly shorter at 60% light irradiance  than at 40% , thus, representing a much more important difference within the hybrid set  as 60.01 μmol·m−2s−1 at 100% light intensity. Besides, it was found that with the decrease in light intensity from 100% to 20%, the light compensation point (LCP) of chrysanthemum hybrid plants was also reduced to the minimum value of 31.43 μmol·m−2s−1 by G × R hybrids, and the difference between treatments and control was significant (n), stomatal conductivity (Gs), intercellular CO2 concentration (Ci), and transpiration rate (Tr) were increased initially from treatment A to C, followed by a gradual decrease in E in all hybrids, indicating that the photosynthetic function in leaf was enhanced with a sudden decline afterwards (s) was increased rapidly from treatment A to D in all F1 hybrids ; 37 × G produced indigo flowers (G: yellow and 37: light purple) a–c.CmSVP gene with the 700 bp target sequence. Among the progeny of G × R transformants, the transgene was detected in five of six strains that were obtained by crossing CmSVP overexpressed plants (G) as a seed parent, while among the progeny of 37 × G, the transgene was detected in four of five strains that were obtained by crossing CmSVP overexpressed plants (G) as pollen parent as shown in CmTFL1 gene expression was interfered in RNAi mediated plants, so F1 hybrids of 37 × R were non-transgenic. To check DNA quality, the endogenous chrysanthemum actin gene was amplified and confirmed for every CmSVP gene-negative plant. These results verified the presence of a transgene in some of the progeny and further demonstrated that the PCR-positive strains were F1 hybrids when one of the parents was transgenic.PCR analysis was performed to amplify a segment of the Tetrastigma hemsleyanum [Dieffenbachia longispatha, well adapted to shade, display a modest capacity for photosynthetic acclimation to increase leaf area [Shading profoundly affects plant development, particularly, characters such as height, internode length, shoot diameter, leaf length and width, color, and shape ,33,34,35eaf area . Leaves are the main organs of plants for the production of photosynthetic assimilates. Impacts of environmental factors on plants or plants’ adaptability to the environment are reflected by the structural attributes of leaves . The intThe biological significance of photosynthesis is to fix carbon and water to synthesize carbohydrates and produce oxygen. Energy derived from photo-assimilates is used for cell division and cell enlargement. It is a well-established fact that reduced photosynthetic activity due to low light intensity hinders plant growth ,50.1 hybrids was found the highest plants exposed to 80% irradiance  is a secondary factor . At the The plants subjected to high irradiance stress typically show lower Fv/Fm values than those of non-stressed plants . Fv/Fm oIt was reported that the Fv/Fm value of high-irradiance stressed plants is comparatively less than non-stressed plants . Photo-iHeavy shading is useful in the reduction of qP , which sCmTFL1 plants of ‘Fenditan’, CmSVP-overexpressed plants of ‘Ganjue’, and non-mutant plants of ‘37′ were grown in the greenhouse of Beijing Forestry University . The optimal conditions for the photoperiod were maintained along with temperatures ranging between 18 °C and 25 °C.The RNAi-mediated CmSVP overexpressed plants (G-♀) × RNAi-mediated CmTFL1 plants (R♂);Chrysanthemum Selection (37♀) × RNAi-mediated CmTFL1 parent (R♂);Non-mutant Chrysanthemum Selection (37♀) × CmSVP overexpressed parent (G♂).Non-mutant Breeding program was organized in accordance with the following 3 crosses:C. morifolium RNAi-mediated CmTFL1 parent ‘‘R’’ and C. grandiflorum CmSVP-overexpressed parent ‘‘G’’ were subjected to a hybridization experiment with one non-transgenic Chrysanthemum selection (“37”). The tubular florets of G were detached before emasculation, and ligulate flower petals were cut to expose the stigma. To ensure safe crossing, the emasculated inflorescences were enclosed in a paper bag for two days, after which a greater number of pollens from freshly opened flowers of R were transferred to the opened stigma of seed parent G with a brush. After pollination, the flowers were recovered to avoid uncontrolled pollination [v/v) mixture of garden soil (1:1:2 mixture of silt: sand: loam) and vermiculite. Trays were kept under artificial growth chambers with controlled light, temperature, and humidity conditions for seeds germination. At the 5-to-6 leaf stage, seedlings were transplanted into medium-sized cups and placed in a greenhouse, followed by transplantation in large pots after one month.lination . The proA) 100%, (B) 80%, (C) 60%, (D) 40%, and (E) 20%, each treatment with 3 replications. Diurnal variations of light strength quanta were measured using a photo quantum probe of the LI-6400XT portable photosynthetic system   . A totalPlant growth parameters, including plant height, the diameter of the main stem, leaf length and width of the fourth functional leaf, and leaf length-to-width ratio, were determined for all plants of each treatment in three cross combinations G × R, 37 × R, and 37 × G.2 rate (Ci), transpiration rate (Tr), and stomatal conductance (Gs) were measured using an LI-6400XT portable photosynthetic system . All gas exchange measurements were recorded at the third fully expanded leaves of chrysanthemum’s F1 hybrids were taken from the apex to measure all gas exchange parameters under the conditions of CO2 level, PPFD, and air temperature at 400 µmol mol−1, 1000 µmol m−2 s−1, and 25 °C, respectively.The net photosynthetic rate (Pn), intracellular CO2, H2 O, the temperature of air and leaf, flow rate, PAR, etc. Such parameters were used for calculating transpiration rate, photosynthesis rate, and stomatal conductance. For each treatment of light intensity in all crosses, 5 plants were selected, and 2 leaves were measured per plant on average. The data were analyzed statistically by using SPSS12.0.The instrument was used for the measurement of photosynthesis is not a single instrument in itself. It is an assembly of instruments for the measurement of CO−2 s−1,600 kHz) for 30 min, whereas and the maximal fluorescence (Fm) induction was performed at saturated light  for more than 0.8 s. The maximal quantum efficiency of PSII was measured using Fv/Fm, where Fv is the difference between F0 and Fm Fs (steady-state fluorescence yield) and Fm’ (light-adapted maximum fluorescence) were obtained at a light source of 600 µmol m−2 s−1 for 0.7 s (Fm’ ± Fs)/Fm’ and Fv’/Fm’ were used to calculate the quantum efficiency of PSII (Φ PSII) and the efficiency of the excitation capture by open PSII centers, respectively [A chlorophyll fluorometer  was used to determine chlorophyll fluorescence by taking a second fully expanded leaf from the plant apex at 25 °C. The minimal fluorescence of leaves (F0) was measured at a weak light pulse (< 0.1 µmol mectively . Photochectively . Non-phoectively . For eacCmSVP gene 5′-ATGATGGTTAGGGAGAAAGTGC-3′ (CmSVP-F) and 5′-TCAACCTGAGTATGGTAATCCTAAC-3′ (CmSVP-R) were used to amplify a 700 bp fragment of SVP gene. A 549 bp fragment of the chrysanthemum actin gene was used to evaluate the quality of DNA using two primers 5′- TCCTCTTAACCCAAAGGCCAACAGA-3′ (CmActin-F) and 5′-TGAGACACACCATCACCAGAATCCA-3′ (CmActin-R). DNA was amplified by using the protocol as mentioned by [The total DNA was isolated from the leaves of rooted seedlings from three cross combinations by following the protocol of . Two priioned by .1 hybrids from their respective parental plants. Leaf size was measured in terms of length and width. Leaf length was measure from the top to the intersectional point of the leaf and its petiole, whereas width was measured from both ends between the widest part of the leaf perpendicular to the mid-rib by a measuring ruler.A comparison of the size and shape of leaves and flowers was performed to differentiate FCmSVP × RNAi F1 hybrids and 37 × CmSVP F1 hybrids are transgenic chrysanthemum plants and can adapt to mild low-light environments, and when light is less than 40% of natural light, low light affects their normal photosynthesis. Full sunlight is not optimal for various physiological and gaseous parameters of chrysanthemums. Therefore, cultivation and production should adjust the planting density and interproduction density so that the relative light strength control in natural light 80% to 60% is appropriate. Moreover, with the changing patterns and increasing intensity of extreme weather events, sophisticated growing systems are inevitably being well adapted to low light conditions. Therefore, these transgenics can be more successful for low light intensity production systems with enhanced photosynthetic activity and high yield. In our study, we observed a significant increase in leaf area that could be correlated to photosynthetic efficiency and biomass production in chrysanthemum plants. In addition, our current transgenic approach may lay a valuable foundation for breeding strategies such as marker-assisted breeding, gene insertion, and ultimately more sustainable flower production of chrysanthemum.The above results show that the progenies of the crossed


N6-methyladenosine (m6A) is the most prevalent type of RNA modification, and we hypothesized that patterns of m6A-related genes may be useful for estimating risk of lung adenocarcinoma (LUAD). An m6A-related gene set variation score (m6A-GSVS) was generated using RNA-sequencing data from LUAD patients in The Cancer Genome Atlas (TCGA). We investigated the association of m6A-GSVS with stemness, tumor mutational burden (TMB), expression of three immune checkpoints, levels of tumor-infiltrating lymphocytes (TILs), and patient prognosis. We found that m6A-GSVS was higher in LUAD than in healthy lung tissue, and it strongly correlated with stemness and TMB. Activated CD4 + T cells were more numerous in LUAD samples that had higher m6A-GSVS than in those with lower scores. Biological processes and pathways, including “Cell cycle,” “DNA replication,” and “RNA degradation,” were significantly enriched in samples with high scores. Furthermore, m6A-GSVS was an independent prognostic indicator in LUAD. In conclusion, we proposed an m6A-GSVS in LUAD. It is a putative indicator for evaluating the ability to RNA m6A, an independent prognostic indicator and associated with tumor stemness. There are more than 100 posttranscriptional modifications known in eukaryotic RNAs , of whicThe m6A modification is catalyzed by the methyltransferase complex, also termed a “writer” , while dAberrant m6A modification has been associated with lung cancer, the most commonly diagnosed cancer and the leading cause of cancer-related deaths . Non-sma1 using the “TCGAbiolinks” package (2. Gene IDs were converted based on the annotation file (gencode.v33.annotation.gff3), downloaded from GENCODE3. Profiles of mRNA expression from the three datasets GSE3141 , two eraser genes (ALKBH5 and FTO), and 11 reader genes .RNA-sequencing data (displayed as raw counts), mutation annotations , and clinical data from LUAD patients were downloaded from TCGA package  in R ver GSE3141 , GSE3021 GSE3141 , and GSE GSE3141 , all bas package . If a ge package , comprisp-value  < 0.05. The unregulated m6A-related genes were explored in The Human Protein Atlas (HPA5). In addition, mutations in those genes were extracted from mutation annotation format (MAF) files using the GDCquery_Maf function in the “TCGAbiolinks” package. Mutation frequencies were visualized as a waterfall plot using the oncoplot function in the “TCGAbiolinks” package.To identify differentially expressed m6A-related genes, the expression profiles of genes were compared between LUAD tissues and healthy lung tissues in the LUAD dataset from TCGA using the “DESeq2” package . ResultsAccording to the currently known m6A theory, we conceptualized an “m6A pathway” that is activated by writers and readers and inhibited by erasers: the writers install m6A, erasers delete it, and readers recognize it. Aberrant expression patterns of genes in the pathway are likely to cause abnormal pathway activity. This is analogous to a previous study  in whichm6A-GSVS = “Writer” enrichment score − “Eraser” enrichment score + “Reader” enrichment score.gsva function in the “GSVA” package   was used package , based o package . The par package . The med6. Several studies have linked m6A-related genes with immune responses to immunotherapy and cancer stem cell in various types of cancers , CD274 (PDL1), and CTLA4.The TMB of each sample was calculated as in a previous study . We compMalignant solid tumor tissues comprise not only tumor cells but also tumor-associated normal cells, such as epithelial, stromal, immune, and vascular cells. These cells are thought to have important roles in tumor growth, invasion, and drug resistance . Based o7. Three reference gene sets were used from the Molecular Signatures Database v7.1   using thase v7.1 , 2015: t8 and “survminer” package9. We carried out uni- and multivariate Cox proportional hazards analyses of the training set in order to compare the prognostic value of the m6A-GSVS with that of routine clinicopathologic features.Kaplan–Meier survival curves were compared between individuals with low or high m6A-GSVS using the log-rank method in the “survival” packaget-test. The Shapiro–Wilk test was used to assess whether the m6A-GSVA score was normally distributed. Intergroup differences in continuous variables were assessed for significance using Wilcoxon, Kruskal–Wallis, or unpaired t-tests. All tests were two-sided and considered significant if p < 0.05, unless otherwise stated.All analyses were performed using R version 3.6 (see text footnote 2). Differentially expressed genes were identified using the unpaired METTL3, RBM15, and VIRMA were upregulated in LUAD, while METTL14, METTL16, WTAP, and ZC3H13 were downregulated. Most reader genes were upregulated in LUAD . The two eraser genes, ALKBH5 and FTO, were downregulated in LUAD.In the TCGA-LUAD dataset, we identified multiple m6A-related genes that were aberrantly expressed in LUAD compared with healthy lung tissue . The wriRBM15   and CTLA4  and no correlation with PDCD1 expression  . This supression .The 24 types of TILs in each individual were displayed as a heatmap . The abucis splicing via spliceosome,” and “mRNA export from nucleus”  biological processes significantly enriched in samples with high m6A-GSVS were “RNA export from nucleus,” “RNA 3 end processing,” “mRNA 3 end processing,” “mRNA nucleus” . The topnucleus” . The topnucleus”  significN = 513), univariable Cox analysis showed that higher m6A-GSVS was associated with poor overall survival (OS) . These results were validated using data from 249 patients with LUAD from the GSE3141, GSE30219, and GSE37745 datasets . Since the m6A-GSVS score showed a skewed distribution, patients were divided into groups with high or low score based on median m6A-GSVS. In the TCGA-LUAD dataset, patients with high m6A-GSVS had worse OS (In the training set (worse OS  and diseworse OS  than thoworse OS . Multivaworse OS .Previous studies investigating the aberrant expression of one or two m6A-related genes in cancer  have sugWe found that m6A-GSVS was strongly associated with stemness, consistent with a previous report linking m6A level with stem cell fate . In breaOur analyses showed that m6A-GSVS positively correlated with TMB, and a previous work linked higher TMB with more new transcripts whose protein products act as new antigens . We infeAs higher TMB is associated with better efficacy of anti-immune checkpoint treatment , high m6cis splicing via spliceosome.” High m6A-GSVS was also associated with enrichment in pathways involving “basal transcription factors,” “cell cycle,” “DNA replication,” “RNA degradation,” and the “spliceosome.” These results suggest that the m6A-GSVS may accurately predict m6A activity. In addition, we found that the targets of the transcription factors MYC and E2F were upregulated in LUAD involving high m6A-GSVS, so these two transcription factors may play a crucial role in m6A progression.Consistent with the idea that m6A affects RNA stability and translation , our GSEWe found that the m6A-GSVS was an independent prognostic factor for patients with LUAD: those who had higher m6A-GSVS had worse OS and DFS than those with lower scores. This finding extends studies linking single m6A-related genes to prognosis in various types of cancer . Rather Our understanding of the mechanism of m6A is still far from complete. The techniques of MeRIP-seq and m6A sequencing have commonly been used to map m6A sites in transcriptomes , but theWe propose the m6A-GSVS as an index of m6A activity in LUAD. This score appears to be associated with tumor stemness and may be useful for predicting prognosis.https://portal.gdc.cancer.gov/; https://www.ncbi.nlm.nih.gov/geo/.Publicly available datasets were analyzed in this study. This data can be found here: PS and SM designed the study. HZ, JH, AL, and HQ performed the experiment. HZ and JH collected the data and prepared the manuscript draft. HJ and HQ performed the statistical analysis. All of the authors approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A linear discriminant analysis differentiated cow milk produced from four agroclimatic zones based on stable isotope ratios, and the inclusion of elemental ratios enhanced the discriminating ability.Information on the geographic origin of milk is important in determining quality attributes and for economic gain through building brand value associated with origin. Stable isotope signatures and trace element concentrations have been increasingly used in authentication of milk, though information on the power of such technology in verifying agroclimatic origin in small continents with diverse climatic, environmental conditions, and animal management practice is scarce. Therefore, the main objective of this study was to investigate the possibility of using a stable isotope composition of C, N, O, and H and element fingerprints to determine the agroclimatic origin of milk produced in different agroclimatic zones of Sri Lanka. Stable isotopes ratios of C, N, H, and O, and elemental fingerprints of milk samples were determined by IRMS and ICP-MS, respectively. Significant variations were observed in stable isotope ratios, especially δ Authentication of geographical origin is a rapidly emerging topic in food safety, quality, traceability, and consumer protection. Tools to validate the geographic origin of food including milk are extremely valuable, as the origin is often associated with quality attributes, for instance, free from disease and pollution. Recent scares, such as melamine adulteration of milk powder , have fo13C and δ15N values of the proteins extracted from milk samples originating from four continents, including Australia , Europe (Germany and France), North America (USA), and Asia (China) has provided evidence about their potential as “fingerprints” of the geographical origin of milk [2H and δ13C have been provided as good biomarkers in milk powder to trace back milk powders to their regional origin in a study conducted in New Zealand [13C and δ15N isotopes of milk casein reflect the inherent geo-climatic variation across dairy farms sampled in the south and north islands [Various analytical techniques, including liquid and gas chromatography, isotope ratio and elemental analyses, spectroscopy, DNA, and sensor techniques, have been widely used to authenticate the geographical origin of various foodstuffs . Among t Zealand . Further islands . Several islands ,19.Most of these studies have been carried out by major dairy exporters, such as Europe, New Zealand, Australia, and Argentina with mostly temperate breeds and more uniform and large-scale dairy operations compared to the South Asian region. In South Asia, the dairy industry is mostly comprised of small-scale farmers  with varying management practices from free-grazing to intensive farming. So far limited work has been carried out on investigating the potential of stable isotope ratios and multi-element analysis in determining the origins of milk in the South Asian region.2) located south of India with a varied climate and topography. The cattle management practices in Sri Lanka vary across the country according to the climate, geographic location, crops grown and cropping pattern, availability of grazing land, and genetic make-up of the animals [Sri Lanka is a small island  was added to the skim milk sample to facilitate precipitation of milk casein at its isoelectric point (pH 4.6). Next, the micro-centrifuge tube was vortexed for 5 min and allowed to settle for 10 min at room temperature to facilitate casein precipitation. Finally, the tube was centrifuged at 20,000× g for 30 min at 4 °C to precipitate milk casein. The supernatant containing the whey fraction was transferred to another tube to separate the milk whey fraction. All these fractions  of the samples were stored at −20 °C until lyophilization.Milk samples were prepared based on the method proposed by Feng et al.  and vali13C and δ15N analysis, whole milk, milk fat, milk casein, and whey were measured as 1.0 ± 0.05 mg, 0.5 ± 0.05 mg, 1.0 ± 0.05 mg, and 2.0 ± 0.05 mg into tin capsules, respectively, and encapsulated. The encapsulated samples were placed in a desiccator until the isotope ratio mass spectrometer (IRMS) analysis was performed. Bulk δ13C and δ15N were measured by combustion to CO2 and N2, respectively, using Euro-EA Elemental Analyzer (EuroVector). The isotope ratio of the gases was measured on a Europa 20–20 isotope ratio mass spectrometer  operating in continuous flow mode. The isotopic compositions of the sample gases were normalized and reported against the international scales for carbon and nitrogen, VPDB, and ambient air. Normalization was made by three-point calibration with international standards, USGS 40 , USGS 41 , and EDTA-OAS laboratory standard . Time-based drift correction was calculated by placing laboratory standards (IAEA 153 milk powder) with EDTA-OAS after every 12 samples. Precision was assessed by placing sequential duplicates of the samples after every 10th sample.For δ18O and δ2H analysis, 0.6 ± 0.05 mg of bulk milk samples were weighed into silver capsules which were closed loosely to allow the exchange of water vapor for correction of the effect of exchangeable hydrogen. Capsule bottoms were slightly flattened to keep them standing upright. Samples were left for six days in ambient conditions to achieve equilibrium of exchangeable hydrogen with the lab atmospheric water. Three replicates were prepared for each sample.For δ2H = −40.78 ± 5.2‰, δ18O = 16.81 ± 0.59‰), USGS-53 , USGS 47 , CH7 (δ2H = −99.2‰), and IAEA601  were used as the reference samples. CH7 and IAEA601 were repeated as control samples at each 15th position throughout the batch for drift correction by linear regression against time. Control samples and USGS-53 were prepared on the same day in a separate tray. Hydrogen and oxygen isotope ratios of the samples were determined by pyrolysis to H2 and CO gases in a Thermal Conversion Elemental Analyzer , followed by isotope analysis of the gas using Thermo Delta-V IRMS in continuous flow mode. Raw results were corrected to the international VSMOW (Vienna Standard Mean Ocean Water) isotope scale using two-point calibration. The measurement uncertainty was calculated by analyzing six to eight replicates of the quality control standard interspersed regular intervals (after every 12 samples) in every batch of samples analyzed throughout the sample analysis period. The measurement uncertainty reported herein is the highest uncertainty value obtained for a single batch of samples throughout the entire sample analysis. The uncertainty values were ±0.18‰ for δ13C, ±0.25‰ for δ15N, ±0.32‰ for δ18O, and ±1.46‰ for δ2H.Samples were placed into the autosampler carousel. Spaces were left for polymer standard materials . The carousel was placed in a desiccator and evacuated to remove air. Samples were vacuum dried on a hot plate set to 40 °C for four days. IAEA Milk Powder 153  for carbon (δ13C), atmospheric N2 (Air-N2) for nitrogen (δ15N), and VSMOW for δ18O and δ2H.The isotopic difference between the sample and an international standard reported in delta notation (δ) units in parts per thousand (‰) is determined by the formula :(1)δ  and 1 mL of H2O2  at 200 °C for 10 min in a MARS-6 Microwave digester  equipped with an EasyPrep Plus high-pressure vessel system. The resulting solutions were then diluted to 25 mL with deionized water (<0.055 µScm−1) and stored in polypropylene bottles at 4 °C until analysis.An aliquot of 1 mL of each milk sample was digested with 5 mL of supra-pure HNO−1 AccuStandard®  stock solution was used to calibrate the instrument. Internal calibration was carried out using 100 µg L−1 of 103Rh and 185Re standards. All measurements were conducted at least in triplicate, and analytical results were expressed as mean values. Analytical quality controlling was assessed using Certified Reference Material BCR-063R . The given reference values and test values showed that the recoveries of available elements ranged from 85–115%, and relative standard deviations by sextuplicate analyses of elements were less than 10%. Further, synthetic external standards, duplicates, and reagent blanks were analyzed at every 10th sample and measurement uncertainties were always less than 2%. Deionized water blanks were also analyzed recurrently to check for sample carry-over.Digested samples were filtered through 0.45 µm cellulose acetate membrane filters, and concentrations of trace elements were measured using Thermo ICapQ high-resolution ICP-MS . Before the analysis, the instrument was optimized with a solution containing Ba, Bi, Ce, Co, In, Li, and U to cover the entire mass range and obtain high sensitivity. A series of multi-element standards prepared by diluting 10 mg Lp-value of ≤0.05 to discriminate samples according to agro-climatic zones based on the elemental composition and isotope ratios as well as isotopic data alone. The predictive ability of the LDA model was evaluated by leave-one-out cross-validation.Results of isotope ratios and elemental concentrations were statistically evaluated using R-Studio version 3.3.0 and SAS . Data were preliminarily explored by preparing box plots and scatter diagrams. Descriptive statistics were taken for each data set. One-way analysis of variance (ANOVA) was used to assess stable isotope data to evaluate whether the variables used differ between the agroclimatic zones, the sampling period’s effect, and their interaction. The means were separated using Duncan’s multiple range tests at a confidence level of 95%. Principle component analysis (PCA) was performed to check if the combined isotopic parameters could be used to distinguish different agroclimatic zones. The linear discriminant analysis (LDA) test was performed with a 13C, 11.6 to 3.3‰ for δ15N, −48 to −155‰ for δ2H, and 26.0 to 16.7‰ for δ18O. The mean δ18O of whole milk samples showed a significant difference among the four agroclimatic zones  rains known to have characteristic isotopic signatures [The present study showed a significant correlation of δaltitude , and a lpitation . The milgnatures . The isognatures .18O values in plants and animals. In Sri Lanka, the temperature decreases at a steady rate of about 6.5 °C for each 1000-m rise. Higher temperatures are experienced generally in the dry zone areas of northern, northcentral, and eastern regions of the island that range between 33.3–34.7 °C [Higher evapotranspiration rates in the dry zone may also lead to enriched δ–34.7 °C . The dry–34.7 °C . 15N values of whole milk, milk casein, and whey revealed a moderate potential for discriminating cattle farming zones. However, this study found that δ15N as well as δ13C values of dry zone samples were significantly different from other zones  and the other zones showed a higher temperature range (27–34 °C) during sampling. According to literature, ambient temperature could influence casein δ15N values [In the present study, the δer zones . The higN values . The iso15N values of milk whey were more depleted than that of milk casein and whole milk in all four agroclimatic regions. A similar observation has been reported by Kornexl et al. [15N value in agricultural products is influenced by the differences in soil conditions, amount of rainfall, soil age, nitrogen turnover rate in soils, and other complex parameters [15N value of milk may reflect the differences in feeding regimes depending on the regional dairy farm [15N with decreasing rainfall [Complete milk protein is composed of 20% whey protein and 80% casein. δl et al. . This fil et al. ,32. Furtrameters ,31. Regiiry farm . There irainfall .13C reflects the relative amounts of C3 and C4 plants used by the regional feeding regimes. According to the Calvin cycles in photosynthetic CO2 fixation, C3 plants show δ13C values between −24 and −32‰. Plants following the C4-dicarboxylic acid pathway show δ13C values between −10 and −19‰ [Brachiaria and Panicum to feed cattle, which fall into the C4 plant category. Gliricidia sepium, which is in the C3 category, was fed to cattle of all the zones except the up country. In up country, Ryegrass (Lolium multiflorum) was given to the animals which is also a C3 plant. However, a considerable amount of concentrate feed, such as rice bran, rice polish, coconut poonac were given as cattle feed in most of the farms. Cattle reared in dry zone eat various types of plants, while freely grazing on communal grazing lands. Therefore, the δ13C values showed a value in between C3 and C4 plants in cattle feed for most of the samples. However, in this study, the δ2H value displayed weak potential to differentiate milk samples of different agroclimatic zones. The variations in δ18O, δ13C, and δ15N values for each of the agroclimatic zone investigated are presented in The difference in δand −19‰ . Except 13C, δ15N, δ18O, and δ2H in whole milk and milk components were significantly influenced by agro-climatic zone, these variables were also moderately influenced by the sampling period . The only variable that displayed a significant difference in wet and dry periods for the dry zone was δ15N of milk casein (p < 0.001). Significant variation between sampling periods in the δ15N values of milk casein and whole milk samples collected from coconut triangle and up country was observed. Mean δ15N of whole milk and milk fractions and δ18O showed relatively enriched values for the wet period compared with the dry period.Although δg period  and less15N during wet and dry seasons may have resulted from variations in ambient temperature and feed material [Forage production and availability in Sri Lanka mainly depend on the rainfall. The availability of lush pastures is high during rainy season and forage is scanty during dry season. Due to the scarcity of forage during the dry season, cattle are often fed with different feeding materials, such as silage and concentrate feeds. The difference of δmaterial ,31.18O and δ2H in different months of the year in agroclimatic zones.The topographical features and regional scale monsoons wind regimes dominate the climate dynamics in each agroclimatic zone. Monsoonal, convectional, and depressional rain contribute to the seasonal rainfall in Sri Lanka. The topographical features of the various agroclimatic zones mean they receive rain in different months of the year. The two main monsoonal rains and two inter-monsoon rains have characteristic isotopic signatures . The up The combined isotope parameters obtained from milk samples were evaluated to distinguish agroclimatic zones using multivariate statistics. The first two principal components explained 69% of the total variance .15N, milk casein δ15N, milk whey δ15N, whole milk δ13C, milk fat δ13C, milk casein δ13C, whole milk δ2H, and whole milk δ18O. The efficacy of the model in discriminating milk samples originated in coconut triangle, dry zone, mid country, and up country from the rest agroclimatic regions were 83%, 92%, 89%, and 97%, respectively. After cross-validation, the above percentages were 75%, 92%, 81%, and 91%, respectively. However, clear clustering was not observed in the score plot of PC1 and PC2 . As the Samples collected during the dry period were evaluated for elemental composition and stable isotope ratios. Previous studies have shown that there is a correlation between the elemental profile of animal products and geographic origin. Further, the elemental profiles of milk have shown to be a useful tool for verification of geographic origin ,36,37. T15N, whole milk δ15N, milk casein δ13C, whole milk δ18O, whole milk δ 2H, Li, Al, Cr, Mn, Fe, Co, Ba, and Sr) showed excellent discrimination in samples from the dry zone, coconut triangle, mid country, and up country (100%) even after cross-validation  correctly classified more than 75% of the milk samples to their respective agroclimatic zones after cross-validation. The inclusion of selected elemental concentrations with stable isotope data  of the milk samples enhanced the discriminating power of the LDA model up to 100%. A combination of selected elemental concentrations and stable isotopic compositions analyzed by a chemometric model is a promising tool to verify agroclimatic origin of cow milk besides high variations in climatic conditions and cattle management practices. Further, the data generated in the present study are expected to be used as baseline data to develop a model for non-targeted screening for potential chemical contaminants, such as dicyandiamide (DCD) and adulterants.This study has demonstrated the ability of stable carbon, nitrogen, oxygen, and hydrogen isotope ratios, and multi-element analysis of cow milk to verify the agroclimatic origin of milk in a small tropical island with a diverse climate, environmental conditions, and cattle management practices. This study provides preliminary information on the applicability of stable isotope and elemental content analysis in verifying agroclimatic origin of milk, even within a small geographic area with diverse conditions. The isotope signatures significantly varied in accordance with the agroclimatic origin of the milk samples and were moderately influenced by the sampling period, detailed data can be found in the


Long working hours have been presumed to negatively influence health. However, evidence is lacking regarding any associations of working hours with depressive mood or suicidal ideation. We investigated the relationships of working hours with depressive mood and suicidal ideation in a representative sample of the Korean general population. We analyzed data collected by the Korea National Health and Nutrition Examination Surveys VI and VII (2013–2018). Depressive mood and suicidal ideation were identified through self-reporting. We divided participants into four groups according to weekly working hours: 30–40, 41–50, 51–60, and > 60 h/week. Sampling weights were applied to obtain estimates for the general Korean population. We analyzed 14,625 participants, of whom 5383 (36.8%), 4656 (31.8%), 2553 (17.5%), and 2033 (13.9%) worked 30–40, 41–50, 51–60, and > 60 h/week, respectively. In these groups, 3.6%, 4.4%, 5.2%, and 6.3% of the participants reported depressive mood, while 1.8%, 1.9%, 2.2%, and 3.6% reported suicidal ideation. In multiple regression analyses, compared with the 30–40 h/week group, the adjusted odds ratios of the 41–50, 51–60, and > 60 h/week groups for depressive mood were 1.35 (1.08–1.69), 1.5 (1.14–1.97), and 1.6 (1.19–2.14). A similar trend was evident for suicidal ideation . Long working hours are significantly associated with depressive mood and suicidal ideation. However, approximately one-third of the world's workforce still works more than 48 h per week2. Among Organization for Economic Co-operation and Development (OECD) member countries, Korea has the third longest working week worldwide (after Mexico and Costa Rica)3. In 2019, Korean working hours were 1967 annually, thus 241 h more than the mean of 1726 h among the 35 OECD countries.Long working hours are associated with negative health effects; many countries worldwide seek to limit working hours. In most European countries, the legal limit is fewer than 48 h per week; approximately half of all European countries impose a 40-h limit6. Long working hours also affect mental health, such that they cause increasing fatigue and distress7. Such negative effects are manifested in several manners, including depressive mood and suicidal behavior that can cause injury and hospitalization, thereby imposing societal burdens of billions of dollars9.Long working hours are closely associated with hypertension, diabetes, cardiovascular disease, and stroke9. Various efforts have been made to explore the psychological processes in the development of suicide ideation and suicidal behavior12. The integrated motivational-volitional (IMV) model of suicidal behavior provides an explanation of the development of suicidal ideation. Suicidal ideation and intent are described as occurring when a person feels that he or she is subjectively trapped in a hopeless situation (entrapment)11. Entrapment consists of a two-dimensional construct: an external entrapment describes external conditions  from which one feels trapped and wants to escape, and an internal trap describes one's own internal limitations and painful thoughts that one feels unable to escape13.Suicidal ideation is one of the strongest predictors of suicidal death and suicide attempts can have negative consequences such as injury and hospitalization, even if it does not end in death16. Several studies found that long working hours have negative effects on mental health , but only a few were large-scale studies using nationwide databases18. Mental health problems are also affected by socioeconomic and environmental factors, which must thus be considered20. Therefore, using a nationwide survey database that includes socioeconomic and environmental factors could be of great advantage. A previous study using nationwide survey data in Korea reported that long working hours were linked to suicidal thoughts18. However, the socioeconomic and environmental factors such as education level, perceived health status, and comorbidities related to suicide were not considered in the study22. We considered potential confounders including social demographic factors and health status when examining the relationships of working hours with depressive mood or suicidal ideation in a large sample of Korean adults.Because workplace problems can act as an external entrapment that can generate suicidal ideation, there have been many studies on the association between work environment and suicidal behaviorOur primary purpose was to examine the associations of working hours with mental health problems, such as depressive mood (sadness or hopelessness) and suicidal ideation (seriously considered), in adults ≥ 19 years of age. Our secondary purpose was to determine the associations of mental health with working hours in subgroups stratified according to sex, shift work status, and occupation type .23; (3) working for fewer than 30 h per week (such people are not regarded as full-time employees in Korea)24; or (4) non-completion of the working hours and/or job questionnaire(s).This cross-sectional study used the databases of the 6th and 7th National Health and Nutrition Surveys (KNHANESs) conducted by the Korea Centers for Disease Control and Prevention (KCDC) from 2013 to 2018. The KNHANES has been performed annually since 1998 to evaluate the health and nutritional status of the Korean population. A national representative sample is obtained using a stratified, multistage cluster-sampling design. Participants vary among years and are not continuously monitored. The survey includes approximately 10,000 independent samples from 192 primary sampling units (PSUs) every year; each PSU is selected based on administrative districts and housing types, using a sampling frame of all census blocks containing the Korean resident registration addresses. The survey features a health interview, health examination, and nutritional survey. In this study, workers ≥ 19 years of age were analyzed. The exclusion criteria were (1) age under 19; (2) unemployed 24.Working hours were determined by asking: “What is your average number of hours worked per week?” Overtime work was included and mealtimes were excluded. The answers were divided into 10-h increments: 31–40, 41–50, 51–60, and > 60 h per weekDepressive mood was assessed by asking “Have you felt sadness or despair which hindered everyday life consistently for 2 weeks or more during the last year?” (yes or no). Suicidal ideation was assessed by asking “Have you ever seriously considered suicide in the last year?” (yes or no).25. Occupational types were classified as white-collar ; pink-collar ; and blue-collar 26. Shift work status was identified. Any major comorbidity such as hypertension, diabetes, dyslipidemia, stroke, myocardial infarction, angina, or a malignancy  was recorded.Demographic characteristics, socioeconomic status, medical histories, and lifestyle habits revealed in the health interview and examination were used in the analysis. The body mass index was the weight divided by the height squared. All participants were divided into non-smokers, ex-smokers, and current smokers. Alcohol consumption was divided into none, ≤ 1 drink/month, 2 drinks/month to 3 drinks/week, and ≥ 4 drinks/week. Educational level was divided into elementary school, middle school, high school, and college or university. Household income level was divided into quartiles. Marital status was divided into single, married, separated, separated by death, and divorced. Perceived health status was recorded as very good, good, average, bad, and very bad, depending on answers to the question “How do you perceive your health?”. Sleep duration was explored by asking “How many hours do you sleep each day?”. Weekly, aerobic physical activity was categorized by intensity: mid-intensity for ≥ 2 h 30 min, high-intensity for > 1 h 15 min, or a combination of mid- and high-intensity activity for a longer time than stated above p-value < 0.05 was considered to indicate statistical significance.Analysis of variance was used to compare continuous variables; the data are presented as means with standard deviations. The chi-squared test was employed to analyze categorical variables; the data are presented as numbers with percentages. We used logistic regression to calculate odds ratios (ORs) with 95% confidence intervals (CIs). We constructed three models to explore potential mediators and confounders. Model 1 was unadjusted. Model 2 was adjusted for age and sex. Model 3 was fully adjusted for age, sex, smoking status, alcohol consumption, educational level, household income, marital status, perceived health status, occupation, sleep duration, physical activity, and comorbidities. We performed subgroup analysis according to sex ; shift work status (no or yes); and occupation . As KNHANES data were derived from multistage complex probability sampling, population weights were applied to all data to ensure that they were representative of the entire Korean population. All statistical analyses were performed using IBM SPSS Statistics for Windows, version 26.0 . A two-sided KNHANES VI and VII were approved by the KCDC Institutional Review Board . Informed consent was obtained from all participants. The study adhered to the principles of the Declaration of Helsinki. All study procedures followed relevant guidelines and regulations.In total, 47,217 people were surveyed. The following individuals were excluded: 9794 aged < 19 years, 13,362 who were unemployed, 4995 who were working fewer than 30 h per week, 4434 who did not report working hours, and 47 who did not report occupations. We finally analyzed 14,625 people . The rates of suicidal ideation also differed significantly among the four groups: 96 (1.8%), 86 (1.9%), 55 (2.2%), and 74 (3.6%) (p < 0.001).The 14,625 participants were divided into individuals who worked 31–40 , 41–50 , 51–60 , and > 60 h/week . Participant demographic and general characteristics are shown in Table p = 0.022), 1.5 , and 1.8 , respectively. Compared with participants who worked 31–40 h, participants who worked > 60 h had a higher OR  for suicidal ideation. In Model 2 (adjusted for sex and age), the ORs for depressive mood of the 41–50-, 51–60-, and > 60-h groups were higher than the OR of the 31–40-h group: 1.42 , 1.75 , and 2.03 . Compared with participants who worked 31–40 h, participants who worked 51–60 and > 60 h had higher ORs for suicidal ideation: 1.59  and 2.7 , respectively. In Model 3 , the ORs for depressive mood of the 41–50-, 51–60-, and > 60-h groups remained higher than the OR of the 31–40-h group: 1.35 , 1.5 , and 1.6 . A dose–response relationship was evident between increased working hours and the risk of depressive mood. Compared with participants who worked 31–40 h, participants who worked > 60 h  exhibited the strongest association with suicidal ideation (Table In Model 1 (unadjusted), the ORs for depressive mood of the 41–50-, 51–60-, and > 60-h groups were higher than the OR of the 31–40-h group: 1.29 . For non-shift work, longer working hours were strongly associated with depressive mood . In terms of occupation, longer white-collar working hours were associated with higher risks of depressive mood . Blue-collar work > 60 h was strongly associated with depressive mood  . In the group working > 60 h, shift work featured a larger OR than did non-shift work (3.02 [95% CI 1.24–7.34] vs. 2.22 [95% CI 1.41–3.49]). Among occupations, blue-collar work > 60 h was associated with the highest risk of suicidal ideation (OR 3.54 [95% CI 2.07–6.06]) , evaluates a patient's suicidal ideation on one of four scales; 0 ("I have no intention of committing suicide), 1 , 2 ("I would like to commit suicide), and 3 (I would commit suicide if I had the chance"). In the KNHANES data we used, the question was asked about seriously considered suicidal ideation, so it could be considered to be equivalent to a level of 2 or higher on the BDI-II scale. A study of 6891 psychiatric outpatients by GK Brown et al. found that those with a BDI-II score of 2 or greater were 6.9 times more likely to commit suicide than those with less than score of 231.Suicidal ideation is defined as any thoughts, images, beliefs, voices, or other cognitions reported by the individual about intentionally ending his or her own life32. The IMV model consists of three phases: a pre-motivational phase (background factors), a motivational phase , and a volitional phase . The pre-motivational phase explains that background factors such as socioeconomic and environmental factors influence suicidal behavior33. In the motivational phase, it was argued that defeat and/or humiliation from which there is no perceived escape—a sense of entrapment is involved in the psychological processes for suicidal ideation to occur. In this process, it was explained that the presence or absence of threat to self-moderators  and motivational moderators  influence the development of suicidal ideations. In our study, we considered background factors  as much as possible and tried to investigate the effect of long working hours, which could act as entrapment, on suicidal ideation.A recent study suggested new risk factors related to suicidal ideation through the IMV model of suicidal behavior36. Long hours also increase anxiety and depression37. Among United Kingdom public officials, individuals who worked more than 55 h were 1.66-fold more likely to have depression and 1.74-fold more likely to experience anxiety, compared with individuals who worked 35–40 h38. Similarly, in our study, the OR for depressive mood was 1.6-fold higher (95% CI 1.19–2.14) for participants who worked > 60 h, compared with participants who worked 31–40 h. Long working hours are also associated with suicidal risk; Kim et al. reported that suicidal ideation was 1.38-fold higher among individuals working > 60 h than among individuals working 40–51 h39. Yoon et al. reported an increased risk of suicidal ideation among individuals working ≥ 60 h compared with individuals working < 52 h18. In our study, compared with participants who worked 31–40 h, the OR for suicide was 2.3-fold greater for participants who worked > 60 h (95% CI 1.54–3.45).Long working hours create distress and affect physical health because of their relationships with lack of exercise, more smoking, and more alcohol consumption41. We found that women who worked > 60 h exhibited a higher OR for depressive mood, compared with men. However, we found a relationship between suicidal ideation and working hours only in men. Shift work causes sleep problems because it does not synchronize with the 24-h circadian rhythm, thereby worsening mental health42. We found similar results. Among shift workers, participants who worked > 60 h exhibited a higher OR for suicidal ideation, compared with non-shift workers. In terms of occupation, Kim et al. reported that blue-collar workers exhibited higher depression scores than did white-collar workers43. In our study, the OR (3.54) for suicidal ideation was highest for blue-collar participants who worked > 60 h.Previous studies have shown that long working hours affected mental health more among women than among men44. Blue-collar workers tend to be more stressed due to conflicts caused by problems such as vertical communication with managers45. Also, blue-collar workers show a more vulnerable health condition than white-collar workers, because they are much exposed to dangerous physical conditions such as hard physical work, noise, and chemicals, and tend to have relatively little interest in health49. Stress and physical illness are well known as major risk factors for suicidal behavior50.There are several possible explanations for blue-collar workers being more suicidal. Blue-collar workers are usually employed as one of the non-managerial occupations, and thus often have low autonomy and decision-making rightsIn our study, the relationship between pink-collar occupation and suicidal ideation showed opposite results depending on whether environmental factors were adjusted or not. In model 1 (unadjusted) and model 2 (adjusted by age and sex), pink-collar occupation was positively related with suicidal ideation, whereas in model 3  it showed a negative relationship. This is because environmental factors such as smoking status, alcohol consumption, educational level, household income, marital status, perceived health status, sleep duration, physical activity, and comorbidities acted as mediators or confounders.51. Second, long working hours reduce sleep quality and volume; sleep problems are associated with depression and anxiety53. Furthermore, if a worker cannot control the workload (and thus may work overtime), an imbalance between effort and reward may develop. Stress increases if the work reward is perceived to be inadequate54. In this way, workers who work long hours may feel defeated and humiliated, with their self-moderators keep threatened, and eventually fall into entrapment55.How do long working hours affect mental health? First, home rest time is shortened; workers do not fully recover from work-induced stress and fatigue56. Fourth, as the questionnaire investigated the mental health of the past year, the specific point in time of the depressive symptoms or suicidal ideation was not clarified. Fifth, depressive mood and suicidal ideation were defined through yes/no dichotomous questions. Though various symptoms such as sadness, hopelessness, fear of the future, and the persistent thought that life is not worth living can indicate depressive mood, the questionnaire used in KNHANES data asked only the sadness or hopelessness57. Suicidal ideation may also vary in severity or definition depending on the measurement method30. Sixth, because ethnic and geographical characteristics may have affected the results, caution is needed when generalizing the results to non-Korean populations. A well-designed large-scale study is required.The principal strength of this study is the very large and nationally representative sample used for analysis. The results can be generalized to the entire adult population of Korea. To the best of our knowledge, this is the largest study thus far regarding the relationships of working hours with depressive mood and suicidal ideation in an Asian population. However, this work had several limitations. First, the KNHANES data were not collected for this study's purposes, so we were not able to go through the rigorous data collection strategies commonly implemented in established research studies. Second, because the study was cross-sectional in nature, we could not infer any causal relationship. Third, because all data were self-reported, recall bias may have influenced the analysis. For example, the number of working hours was measured via a single self-reported question. Though self-reported questionnaires had been proven useful in examining working hours at the population level in several studies, more objective measurements will be needed in further studiesWe studied a nationally representative Korean population. After adjustments for sociodemographic and mental health-related variables, we found that long working hours were strongly associated with depressive mood and suicidal ideation. Men, as well as shift and blue-collar workers, were more vulnerable to suicidal ideation. Working hours should be limited by law.


This data article is related to a research paper entitled ``Correlations between spectroscopic data for charge-transfer complexes of two artificial sweeteners, aspartame and neotame, generated with several π-acceptors'' [J. Mol. Liq. 333 (2021) 115904]  Specifications Table•Investigating the charge-transfer (CT) properties of the artificial sweeteners, aspartame and neotame may be useful toward improving their uses and applications.•Verifying the stoichiometry of the interaction between the artificial sweeteners, aspartame and neotame with different acceptors is very important to understand the mode of the interaction of these sweeteners toward improving their uses and applications.•The most important and useful methods to determine the stoichiometry of the interaction between molecules are Job's continuous variation method and the spectrophotometric titration method. These methods are easily performed.1−4 M were each individually mixed with three acceptor solutions in methanol solvent at the same concentration (5 × 10−4 M). These acceptors were CA, PA, and DDQ. The resultant donor-acceptor systems were Asp−CA, Asp−PA, Neo−DDQ, Neo−CA, and Neo−PA, which were scanned using a UV/Vis spectrophotometer (The UV/Vis spectra dataset were provided as separated Excel sheet). The stoichiometry of the interaction between the donors (Asp and Neo) and the acceptors  was obtained using i) Job's continuous variation method Methanolic solutions of aspartame (Asp) and neotame (Neo) as donors at concentration of 5 × 1022.114H18N2O5; 294.30 g/mol; purity ≥ 98%), Neo  were bought from SUPELCO Analytical Company. PA , CA , and DDQ  were bought from Sigma-Aldrich Chemical Company.Analytical-grade Asp , and acceptors  solutions were individually prepared at 5 × 102. The solubilized donor (Asp or Neo) (1 mL) was combined with each solubilized acceptor  (1 mL) and methanol (3 mL) in 5-mL glass tubes to generate the Asp−CA, Asp−PA, Neo−DDQ, Neo−CA, and Neo−PA systems.CT).3. The UV-visible spectra of the free compounds and prepared systems were collected at room temperature from 200 to 800 nm using a Perkin−Elmer Lambda 25 UV/Vis spectrophotometer and used to obtain the CT band (λCT) of 10 standard solutions with varied donor to acceptor molar ratios (from 4:1 to 1:4) were plotted against the volume of the acceptor in each standard solution.4. To verify the stoichiometry of the interaction between donors and each of the acceptors using the spectrophotometric titration method, the absorbances (λCT) of 10 standard solutions with varied molar fractions of donor and acceptor (Cdonor + Cacceptor) were plotted against the molar fraction of the acceptor in each standard solution.5. To verify the stoichiometry of the interaction between donors and each of the acceptors using Job's continuous variation method, the absorbances (λAbdel Majid A. Adam: Data curation, Writing – original draft, Writing – review and editing; Tariq A. Altalhi: Conceptualization, Methodology, Software, Supervision; Hosam A. Saad: Visualization, Investigation, Software, Validation; Amnah M. Alsuhaibani: Visualization, Investigation, Software, Validation; Moamen S. Refat: Conceptualization, Methodology, Software, Supervision; Mohamed S. Hegab: Visualization, Investigation, Software, Validation.The authors declare that they have no known competing financial interests or personal relationships that could be perceived to have influenced the work reported in this article.


N = 1604), participants asked to imagine that they had recovered from COVID‐19 reported lower social distancing intentions compared to a control condition. Participants who suspected (versus imagined) that they had recovered from past infection did not report lower preventative intentions compared to the control condition, even at high levels of certainty of past infection. In Study 2 (N = 1732), introducing the idea of immunity passports also reduced social distancing intentions compared to a control condition. The latter effect was, however, attenuated when cautious information about the equivocal science on COVID‐19 was also presented to participants. Participants who suspected that they had COVID‐19 in the past (compared to the control condition) revealed a similar pattern of results, but only at higher levels of certainty of past infection. Caution regarding infection‐acquired COVID‐19 immunity and immunity passports will be crucial in the COVID‐19 response. Implications for premature pandemic announcements, as well as their potential remedies, are discussed.Research suggests that emerging information about infection‐acquired COVID‐19 immunity should be interpreted with caution. The introduction of “immunity passports” that would enable people who have recovered from COVID‐19 to travel freely and return to work may therefore have detrimental consequences if not managed carefully. In two studies, we examined how perceived (suspected or imagined) recovery from COVID‐19, and the concept of immunity passports, influence people’s intentions to engage in behaviors aimed to reduce the spread of COVID‐19. We also consider ways to lessen potential negative effects. In Study 1 ( That means I can’t get it (immune), and can’t give it. Very nice to know!!!”– Tweeted by Donald J. Trump, 11/10/2020.Governments around the world have suggested issuing “immunity passports” to people who have recovered from COVID‐19.1.1Early on during the pandemic, many optimistic headlines gave the impression that it is unlikely for people to be infected with COVID‐19 more than once . In Study 2, we focused on the effects of being exposed to the concept of immunity passports, and incautious versus cautious information regarding immunity, on social distancing, hygiene, and face‐covering intentions. Again, we also examined the effect of suspected COVID‐19 status, with certainty of said status as a potential moderator.In two studies, we examined the effects of perceived recovery, and information about immunity passports, on intentions to mitigate the spread of COVID‐19. In Study 1, we examined the effects of https://www.osf.io/6qtmy/registrationsIn both studies, we recruited participants by means of convenience sampling. Study 1 was advertised on three social media platforms: Facebook, Twitter, and Reddit (the majority of participants were recruited from the latter), and Study 2 was solely advertised on Reddit. In all cases, the same advertisement was posted on each platform, referring to a survey on COVID‐19 that was being conducted by psychology researchers at the University of Kent. No incentives were offered. Our efforts were mainly focused on Reddit for several reasons: (1) Reddit now boasts 52 million daily active users worldwide Reddit, , with ju2imagined they had recovered from COVID‐19 would show lower COVID‐19 preventive intentions  not tested and do not suspect to be currently infected; b) not tested and do not suspect to be currently infected, but suspect to have had it in the past; c) not tested, but suspect to be currently infected, d) have been tested and results were negative; e) have been tested, results were positive and they were still infected; f) have been tested, results were positive but they had since recovered; and g) have been tested, results were positive but they were unsure of their recovery status. All participants completed the dependent measures, but in Study 1, only group a were assigned to the experimental conditions, and group b was used as a comparison group for the moderation analyses.Finally, we predicted participants who 2.12.1.1d = 0.20) with a power of 0.80, at least 394 participants were required in each experimental group. We recruited 1768 participants from 13th to 14th May 2020 via posts on social media  and Reddit forums. Vaccines against COVID‐19 were not available at this time. Participants who suspected that they do not currently have COVID‐19  and those who suspected they had it in the past (group b: n = 426) were included in the analyses, and the remaining participants were excluded (n = 137).n = 28). The remaining participants  were included in the final analyses. Of these participants, 65.1% were American, 22.4% were UK nationals, and the remaining 12.5% were made up of 58 different nationalities; 4% were frontline healthcare workers and the remaining 96% were not; finally, 30% had an underlying health condition, 57% did not, and the remaining 13% were not sure.G*Power determined that in order to detect a small effect  and group b, are moderated by certainty of suspected COVID‐19 status.The first part of the study was experimental, including three conditions , consisting of participants from group 2.1.2yes or no). Participants who reported yes were then asked what the result of their test was (positive or negative). Participants who reported positive were then asked about their current condition . Participants who reported no to being tested were then asked whether they suspect they currently had COVID‐19 , and how certain they are of this .Participants were asked questions about their current COVID‐19 status. They were asked if they have been tested for COVID‐19 . In both conditions, participants were then asked to try to put themselves in the shoes of someone with their imagined COVID‐19 status and to think about how this would make them feel. Participants in the control condition were not presented with any information.Participants from group Not at all worried, 7 = Extremelyworried).not at all, 7 = very much), and to describe them in a textbox.All participants were then asked to report how worried they were about COVID‐19  apart from other people”; α = 0.87) and hygiene  intentions over the next month , and whether they work in frontline healthcare (yes or no), before being debriefed and thanked.Finally, all participants completed several measures that were included as covariates; age, gender, education  control versus (1) “infected”; X2 = (0) control versus (1) “recovered”. Social distancing and hygiene intentions were entered as dependent variables. All covariate measures were included.d = 0.14), and “infected” significantly increased (Cohen’s d = 0.93), social distancing intentions. Only “infected” significantly increased (Cohen’s d = 0.29) hygiene intentions. Of the covariates, age, gender , and underlying health conditions (yes) positively predicted all outcomes.The regression models were significant and accounted for 18% and 5% of the variance in social distancing and hygiene intentions respectively Table . Compare2.2.2suspected COVID‐19 status (group a versus group b) predicted worry about COVID‐19, social distancing, and hygiene intentions, and whether these relationships were moderated by certainty of suspected COVID‐19 status, we performed a series of hierarchal regression analyses. We created a dummy coded variable representing suspected COVID‐19 status: 0 = group a versus 1 = group b. For all dependent variables, Step 1 tested the main effects of suspected COVID‐19 status (predictor) and certainty of said status (moderator) and also included all covariates. In Step 2, we added the two‐way interaction of the predictor and moderator. The continuous moderator variable was mean‐centered prior to analyses.To test whether β = −0.07, p = .080; R2 = 0.07, F = 7.48, p < .001), hygiene intentions  = 4.41, p < .001), or worry about COVID‐19  = 15.00, p < .001). Certainty of suspected COVID‐19 status negatively predicted worry about COVID‐19 , but not social distancing  or hygiene intentions . In Step 2, the interaction term did not significantly increase the variance for social distancing  = 0.02, p = .888), hygiene intentions  = 0.07, p = .787), or worry about COVID‐19  = 2.83, p = .093). Thus, we did not find the expected moderation effects.In Step 1, suspected COVID‐19 status did not predict social distancing  in the experimental conditions. However, by splitting the control condition in the current study, this still allowed us to compare group a and b’s COVID‐19 preventive intentions and worry about COVID‐19, and whether these relationships are moderated by certainty of suspected COVID‐19 status.Study 1 demonstrated that All participants in the experimental conditions were asked to imagine that they had recovered from COVID‐19 and were then exposed to the concept of immunity passports. These participants were then split randomly into three conditions where they received: (1) no further information, (2) incautious information about infection‐acquired COVID‐19 immunity, or (3) cautious information about infection‐acquired COVID‐19 immunity. Finally, there was also a control group, wherein participants were not asked to imagine past infection and were not presented with any information about immunity or immunity passports. Relative to the control group, we expected (1) and (2) to indicate incremental reduced behavioral intentions, but that (3) would display similar levels of behavioral intentions.3.13.1.1d = 0.20) with a power of 0.80, at least 394 participants were required in each experimental group. We recruited 1999 participants from 8th to 12th June 2020 via posts on Reddit forums. Vaccines against COVID‐19 were not available at this time. Participants who suspected that they did not currently have COVID‐19  and those who suspected they had it in the past (group b: n = 387) remained and all other participants were excluded (n = 242).n = 24). The remaining participants  were included in the final analyses. Of these participants, 56.6% were American, 27.7% were UK nationals, and the remaining 15.7% were made up of 50 different nationalities; 4% were frontline healthcare workers and the remaining 96% were not; finally, 25% had an underlying health condition, 62% did not, and the remaining 13% were not sure.As in Study 1, G*Power determined that in order to detect a small effect  in behavioral intentions, and worry about COVID‐19, are moderated by certainty of suspected COVID‐19 status.As in Study 1, the first part of the study was experimental, including four conditions , consisting of participants from groups 3.1.2Participants were asked the same series of questions about their current COVID‐19 status as in Study 1, including certainty of suspected status.a and b were then randomly assigned to one of four conditions; “immunity”, “incautious [information about immunity]”, “cautious [information about immunity]”, and a control group. As in Study 1, participants in the “immunity”, “incautious”, and “cautious” conditions were asked to imagine that they have recovered from COVID‐19. Additionally, they were then presented with brief information regarding the concept of “immunity passports” . Participants in the “incautious” condition were further presented with two incautious, and participants in the “cautious” condition were presented with two cautious, statements regarding evidence of immunity . In all three experimental conditions, participants were asked to keep their imagined COVID‐19 status in mind and to let the additional information about immunity sink in. Participants in the control condition were not presented with any information about immunity passports and were not asked to imagine that they had been infected in the past. As in Study 1, all participants were then asked to report how worried they are about COVID‐19 and participants in the experimental conditions only were also asked to report how vivid and clear their thoughts were, and to describe them in a textbox.Participants from groups r = 0.79). As in Study 1, participants in the three experimental conditions were asked to keep their imagined COVID‐19 status in mind when answering these questions. Finally, all participants completed the covariate measures, and were then debriefed and thanked, as in Study 1.All participants then reported their social distancing (α = 0.88) and hygiene (α = 0.63) intentions, as in Study 1. Two new items measured face covering intentions  “immunity” versus (1) control; X2 = (0) “incautious” versus (1) control, X3 = (0) “cautious” versus (1) control. Social distancing, hygiene, and face covering intentions were entered as dependent variables.d = 0.31, Cohen’s d = 0.41, respectively), and “cautious” information had no effect on, social distancing intentions. Only “cautious” information significantly increased (Cohen’s d = 0.11) hygiene intentions. “Immunity” and “incautious” information significantly decreased , and “cautious” information had no effect on, face covering intentions. Of the covariates, age, gender , and underlying health condition (yes) positively predicted all outcomes.All regression models accounted for significant variance Table . Compare3.2.2certainty of suspected COVID‐19 status moderated the effect of suspected COVID‐19 status (0 = group a versus 1 = group b) on social distancing, hygiene and face covering intentions, and worry about COVID‐19.As in Study 1, we performed analyses testing whether β = −0.06, p = .274; R2 = 0.11, F = 6.92, p < .001), hygiene  = 1.15, p = .331), face covering intentions  = 6.01, p < .001), or worry about COVID‐19  = 4.61, p < .001). Certainty of suspected COVID‐19 status negatively predicted social distancing , face covering intentions , and worry about COVID‐19 , but not hygiene intentions .In Step 1, suspected COVID‐19 status did not predict social distancing  = 6.22, p = .013), but not for hygiene  = 0.66, p = .418), face covering intentions  = 0.05, p = .816), or worry about COVID‐19  = 0.39, p = .531). Simple slopes analysis using PROCESS  but was not significant for group a , but were attenuated when cautious information was presented . Furthermore, hygiene intentions were improved compared to the control group when cautious information was presented about immunity. These findings partially support recent research demonstrating that perceived infection‐acquired immunity may be damaging to social distancing and hygiene intentions . Thus, the literature would benefit from replicating the current findings in designs that focus on people who have actually recovered from COVID‐19. Fourth, despite the large sample sizes, participant recruitment relied heavily on Reddit as its main source. Certain demographics  collected in the current studies were comparable to those in research that uses popular crowdsourcing platforms (e.g., Amazon’s Mechanical Turk, Green & Douglas, Thirdly, although 5Governments have suggested introducing immunity passports (McMillan, The authors have declared no conflicts of interest for this article.Supplementary MaterialClick here for additional data file.


The article “Modeling Cash Plus Other Psychosocial and Structural Interventions to Prevent HIV Among Adolescent Girls and Young Women in South Africa (HPTN 068)”, written by Marie C. D. Stoner, Jessie K. Edwards, Daniel Westreich, Kelly Kilburn, Jennifer Ahern, Sheri A. Lippman, F. Xavier Gómez-Olivé, Kathleen Kahn, Audrey Pettifor was originally published electronically on the publisher’s internet portal on 21st January 2021 without open access. With the author(s)’ decision to opt for Open Choice the copyright of the article changed on 9th April 2021 to © The Author’s 2021 and the article is forthwith distributed under a Creative Commons Attribution.“The original article has been corrected.”


Ultrasound, alone or in combination with natural antimicrobials, is a novel food processing technology of interest to replace traditional food decontamination methods, as it is milder than classical sterilisation (heat treatment) and maintains desirable sensory characteristics. However, ultrasound efficacy can be affected by food structure/composition, as well as the order in which combined treatments are applied. More specifically, treatments which target different cell components could result in enhanced inactivation if applied in the appropriate order. The microbial properties i.e. Gram positive/Gram negative can also impact the treatment efficacy.Listeria innocua (Gram positive) and Escherichia coli (Gram negative), at a range of cavitation conditions . The order of treatment application was varied, and the impact of system structure was also investigated by varying the concentration of Xanthan gum used to create the food model systems (0 – 0.5% w/v). Microbial inactivation kinetics were monitored, and advanced microscopy and flow cytometry techniques were utilised to quantify the impact of treatment on a cellular level.This work presents a systematic study of the combined effect of ultrasound and nisin on the inactivation of the bacteria E. coli at 500 kHz only, with L. innocua demonstrating resistance to all frequencies studied. Enhanced inactivation of E. coli was observed for the combination of nisin and ultrasound at 500 kHz, but only when nisin was applied before ultrasound treatment. The system structure negatively impacted the inactivation efficacy. The combined effect of ultrasound and nisin on E. coli was attributed to short-lived destabilisation of the outer membrane as a result of sonication, allowing nisin to penetrate the cytoplasmic membrane and facilitate cell inactivation.Ultrasound was shown to be effective against  Furthermore, the mechanisms of resistance to nisin through modification of the cell wall can lend resistance to antibiotics with a similar mode of action, meaning that typical treatments for listeriosis (for example) may not be as effective Listeria species isolated from the environment have become more prevalent in recent years, which is of huge concern due to the high mortality rates associated with listeriosis The order in which treatments are applied could impact their efficacy and the microbial response, depending on the target in the cell i.e. a treatment which damages the outer membrane followed by one which acts on internal components of the cell could result in enhanced inactivation The chemical composition and rheological/structural properties of liquid and solid(like) foods can vary greatly, even between liquid food products. This can impact the efficacy of ultrasound treatment: bubbles become more stable as the system viscosity increases and are less likely to collapse, potentially affecting the transient cavitational activity Listeria) are more resistant to ultrasound than Gram negative bacteria (e.g. E. coli) The efficacy of ultrasonic cavitation can be impacted by many other factors including frequency, applied power, sonication time, system volume, pressure amplitude, type of transducer i.e. equipment set-up, probe or plate diameter, liquid height and liquid temperature Ultrasound inactivation of microorganisms both alone and in combination with antimicrobials  has been studied in a range of liquid systems such as wastewater, saline solution, and growth medium Listeria innocua (Gram positive) or Escherichia coli (Gram negative), in systems of varied viscosity.This study aims to identify the effect of ultrasound at a range of frequencies, alone and in combination with a sublethal concentration of nisin, on 22.1g in 50 mL falcon tubes  for at least 30 min for removal of entrapped air bubbles . After autoclaving at 121 °C for 15 min, the viscoelastic medium was centrifuged again to remove additional air bubbles.Xanthan gum (XG) was selected as the gelling agent in this study as it is stable at a wide range of temperatures and is widely used in the food industry as a thickener and stabiliser in food products such as dressings, sauces, and bakery items 2.2G’ and loss modulus G” were measured. G’ represents the elastic portion of the material response (somewhat a measure of the stiffness of a gel), while G” represents the viscous response. The loss tangent (tanδ = G”/G’) indicates whether the material is closer to an elastic solid (tanδ < 1), or a viscous fluid (tanδ > 1).The rheological stability of the XG systems was tested at 37 °C, the optimum growth temperature for both species, by conducting dynamic oscillatory measurements to assess the viscoelastic behaviour. More specifically, the frequency dependence and magnitude of the storage modulus G’ and G” values were obtained over a frequency range of 1–100 rad/s using a rotational Physica MCR 200 rheometer , with the temperature regulated using a Paar Physica circulating water bath . A cone and plate geometry was used  and at least two independent replicates of at least two samples were analysed for each viscoelastic system. The strain was kept constant at 2%.2.3L. innocua ATCC 33090, a model for the foodborne pathogen L. monocytogenes, and of E. coli ATCC 25,922 were stored at −80 °C in TSBYE , supplemented with 15% glycerol. A loopful of thawed culture was inoculated in 15 mL TSBYE for 9.5 h at 37 °C, the optimum temperature for growth. 20 µL was subsequently transferred to fresh 15 mL TSBYE and cultured for 15 h at 37 °C until early stationary phase was reached (109 CFU/mL). Each species was cultured to stationary phase separately.Stock cultures of 2.4Interchangeable piezoelectric transducers of 44 kHz, 500 kHz and 1000 kHz (Honda Electronics), comprising a 5 cm diameter piezoelectric ceramic round adhered to a 10 cm diameter vibration plate, were screwed onto the base of a custom-made plastic box (12 × 12 × 16 cm). The contact area between the transducer plate and the liquid carrier was 7 cm diameter. An amplifier  was connected via (i) an impedance matching device (IMD) and (ii) a discharge plug to the transducer. The IMD and discharge plug were used to minimise the reflected power (RP) to the amplifier, alongside frequency tuning as required . The appBorosilicate scintillation vials  were used to hold the biological samples, which contained a constant volume of 10 mL. Any variation between vial position/wall thickness is accounted for in the measured experimental error. The sample bottle was suspended in 500 mL of degassed deionised water inside the reactor vessel, aligned vertically over the centre of the transducer plate at a height of 1.7 cm above the plate, with the liquid level inside the vial aligned with the liquid level in the vessel  to the maximum temperature obtained of 46 °C , thus si2.5pc is the specific heat capacity of water (4.2 J/Kg), M is the total mass of water (g), and dT/dt is the temperature rise per second 3 and the total mass of water was 510 mL i.e. the reactor vessel contents (500 mL) plus the sample vial volume (10 mL).For calorimetric quantification, 10 mL distilled water was used in the place of a biological sample with a temperature probe suspended in the centre of the vial. The system was sonicated at 44 kHz, 500 kHz or 1000 kHz with an applied power of 30 W and the temperature recorded every 30 s for 15 min of sonication. The calorimetric power  was calc- ions are oxidised to give I2. Excess I- ions present in the solution can react reversibly with I2 to produce the I3- ion, which appears yellow in solution: the change in colour can be used to indicate the concentration of I3-. A 0.1 mol/L KI solution was prepared with deionised water. 10 mL KI solution was sonicated at 44 kHz, 500 kHz or 1000 kHz with an applied power of 30 W for up to 15 min. The absorbance of I3- at 355 nm was recorded and the I3- concentration calculated using the Beer-Lambert absorption law  KI dosimetry was conducted as per the method of Koda et al. 2.6L. innocua or E. coli was cultured and grown to stationary phase in 15 mL TSBYE at 37 °C, as previously described 6 CFU/mL. 10 mL of these systems was transferred into a borosilicate scintillation vial. Samples remained at room temperature (approx. 20 °C) for a maximum of 1 h before ultrasonic treatment for 0 – 30 min at either 44 kHz, 500 kHz or 1000 kHz, and were removed immediately to an ice bath for a maximum of 2 h before sample processing. Samples were serially diluted in TSBYE and plated on tryptic soy agar supplemented with 0.6% YE (TSAYE) plates for enumeration of both species. Plates were incubated at 37 °C for 24 h before enumeration (CFU/mL). To maintain a constant sample volume during ultrasound treatment of up to 30 min, samples were sonicated for the required duration  then sacrificed for processing. Each point presented in 2.7L. innocua or E. coli at a cell density of 106 CFU/mL were prepared in 0%, 0.1%, 0.3% and 0.5% XG systems, as previously described for ultrasound inactivation. The nisin treatment time was fixed at 30 min, and was applied either before or after ultrasound treatment, which was varied between 0 and 30 min. Three independent experiments with two replicate samples per experiment were conducted.10 mL samples of L. innocua . This test was not performed on E. coli as nisin is not effective on Gram negative species Nisin was applied at a concentration of 35 IU/mL, which was selected as it resulted in a 1 log reduction of 2.8Solutions of 0%, 0.1%, 0.3% and 0.5% XG containing 1 mM luminol  (Sigma Aldrich) and 0.1 M NaOH (Sigma Aldrich) were prepared for use immediately prior to sonochemiluminescence (SCL) and kept protected from light until use. The sonication equipment was moved into a dark room in order to detect and capture the released luminescence, which is not visible by the naked eye. 10 mL samples were sonicated as previously described for inactivation experiments. An ANDOR iXon3 EMCCD camera, operating at – 70 °C, and software was used to capture images of the systems after 30 s of sonication, with an applied EM gain level of 15 and exposure time of 1 s. Raw images were exported as 8-bit TIFF-files and the intensity value calculated using ImageJ 2.0 2.9Flow cytometry analysis was conducted to obtain an indication of the level of injury to the overall population rather than identifying only the dead cells (i.e. inactivation kinetics based on colony forming unit (CFU) counts on TSAYE), to gain a better understanding of ultrasound mechanisms, and to further characterise the effects of sonication. Propidium iodide (PI) and bis- trimethaine oxanol (BOX)  fluorescent stains were used to visualise the dead population and the injured population respectively. PI enters cells that have lost their membrane integrity (i.e. dead cells) and binds to nucleic acids, giving a bright red fluorescence. BOX enters depolarised cells, i.e. where the membrane potential is reduced to zero, binding to the membrane or intracellular proteins to give a bright green fluorescence. Cell depolarisation is an indication of cell injury Flow cytometry analysis was conducted using an Attune NxT flow cytometer . A 0.5 mL sample was stained with BOX (100 ng/mL) and PI (4 µL/mL) and incubated at room temperature for 5 min. The stained samples were excited using a 488 nm solid state laser and fluorescence was detected using 530/30 BP (BL1 channel) and 620/15 BP (YL2 channel) filters corresponding to BOX and PI fluorescence respectively. Approximately 10,000 data points were collected and the data was analysed using Attune NxT software. Three independent experiments were performed with two replicates each time.2.10g for 10 min at 4 °C. The cell pellet was resuspended in 1 mL sterile Dulbecco’s Phosphate Buffered Saline (DPBS), modified without CaCl and MgCl2, then 1 mL of 3% v/v formaldehyde solution was added to the resuspended cells and incubated for 1 h at room temperature to fix the cells. 3 mL DPBS was added to the total fixed sample to make a total volume of 5 mL.Scanning Electron Microscopy (SEM) was conducted on liquid samples following ultrasound treatment to identify any impact of ultrasound at a cellular level. 10 mL samples were sonicated for 30 min and the cells were collected by centrifugation at 4500 A sterile 13 mm diameter polycarbonate track-etched membrane filter  held in a sterile Swinnex filter holder  was wetted with 2 mL DPBS then the fixed cell suspension was passed slowly through the filter. The filter was removed to a sterile petri dish and dehydrated in an ethanol in MilliQ series  for 10 min at each stage, repeating the 100% stage twice. The samples were air dried overnight and subsequently transferred to a desiccator for 12 h. Filters were mounted onto aluminium stubs using silver paint at each corner and gold sputter coated in a vacuum evaporator . Samples were visualised using a JSM-7100F Field Emission SEM (JEOL Ltd).2.11t-test was used to compare two mean values. Differences were considered significant at P < 0.05. Standard deviation is reported as error bars on the plots.To ensure statistical significance, at least three independent experiments were conducted with two replicate samples throughout. Quantitative results were collected in Microsoft Excel and the mean, standard deviation and standard error were calculated for each replicate result. Student’s 33.1L. innocua and E. coli treated with ultrasound at 44 kHz, 500 kHz, and 1000 kHz, in combination with or without artificial nisin (35 IU/mL) added for 30 min before or after ultrasound treatment, in liquid TSBYE. The combination of ultrasound and nisin will be discussed in 3.1.1L. innocua, no effect of ultrasound is observed at any frequency, although an effect of the added nisin is observed at all frequencies  nisin, (ii) ultrasound  and (iii) high-pressure treatments on Listeria seeligeri in liquid whole egg was studied L. monocytogenes 10403S inactivated in saline solution for 20 min Listeria: for example Muñoz et al. L. innocua treated with ultrasound in a transparent buffer system (no nutrients) , and Inguglia et al. L. innocua treated with ultrasound in TSB  Listeria species. Furthermore, the differences in efficacy reported in the literature are likely due to the variations in power, experimental set-up, treatment time and intensity, and the strain of bacteria used: variations in resistance to ultrasound between strains of Listeria has been reported Listeria species, only that the strain L. innocua ATCC 33,090 (used in this study) is resistant to these settings.For quencies a-c. Morequencies a-c. The 3.1.2E. coli by ultrasound alone is observed at 500 kHz only, i.e. a 1-log reduction in cell count is observed at this cavitation condition and not at the other conditions studied  was observed at 500 kHz only and not at 44 kHz or 1000 kHz  with inactivation at 500 kHz attributed to a combination of physical and chemical effects of ultrasound Sonochemical effects in general are enhanced in the range 200–600 kHz E. coli was observed following ultrasonic treatment at 40 kHz and 600 W, using a bath configuration E. coli and S. aureus was also observed at 30 kHz and 100 W with a horn configuration E. coliThe lack of inactivation at 44 kHz in the present work is attributed to the relatively low power compared to previous studies. Another group using a similar plate experimental set-up also observed no microbial reduction for the treatment of abattoir wastewater at 44 kHz and applied power 40 W It is also noted that erosion of aluminium foil was observed at 43 kHz and 1 W in a plate system, although for an irradiation time of 180 min As noted in the Materials and Methods, the experimental set-up was kept constant at each frequency condition. As such, it is possible that activity at 44 kHz and 1000 kHz might be observed if the set-up is optimised for each frequency applied, however this was out of the scope of this work and will be addressed in our future work (see the Conclusions).3.23.2.1L. innocua at any frequency, regardless of the order of application, as also observed for individual treatments  and nisin. The effect of ultrasound treatment on a cellular level at this frequency is clear from the SEM images: far fewer intact cells are observed for ultrasound treatment  cell envelopes lacking in cytoplasmic content but retaining their morphological and structural features Cellular debris consists of cellular contents that have been released or expelled through the membrane following cell death: the presence and quantity of cellular debris therefore provides an indication of the occurrence and level of cell death. As such, the presence of both ghost cells and cellular debris following ultrasound treatment b suggestE. coli following sonication at 500 kHz (applied power 12.4 W), were also observed by Koda et al. E. coli, L. innocua, S. aureus, blood cells, and yeast Empty cell envelopes of E. coli . Under certain processing conditions, ultrasound can destabilise the outer membrane of E. coli without rupturing the cytoplasmic membrane As such we propose that, in addition to the terminal damage to the cell wall and membrane, ultrasound may also cause short-lived damage to the outer membrane of E. coli by ultrasound  and nisin, added immediately before ultrasound treatment  saw a ∼0.5 log reduction at 35 °C, but ultrasound did not enhance the inactivation effect of nisin, in contrast to our findings Elsewhere the inactivation of 3.3E. coli is studied at 500 kHz only, as inactivation in the liquid system was observed only at this cavitation condition, thus any effect of viscosity (expected to reduce the impact of inactivation) should be visible. Inactivation of L. innocua was not continued in the viscoelastic models, as no effect was observed in the liquid system  reduces as the XG concentration increases, indicating that the system becomes less fluid and more solid(like) as a result  increases, with no effect of ultrasound and/or nisin observed for the 0.5% XG system  to solid(like) exists between 0.3% and 0.5% XG, explaining the lack of inactivation effect in the 0.5% XG system . ViscosiThere are no existing studies which investigate the efficacy of ultrasound in varied systems of controlled composition and complexity with which the present study can be compared. Furthermore, while studies have been carried out in a range of liquids  and liquid foods , the experimental set-up, frequency, microbial species and power intensity varies between them making a useful comparison between systems very difficult 4L. innocua (Gram positive) and E. coli (Gram negative) species was presented. Crucially, the experimental set-up used allowed for a systematic comparison of different cavitation conditions while maintaining all other ultrasonic parameters. L. innocua was resistant to ultrasound treatment at the frequencies studied, while ultrasound was only effective for E. coli at 500 kHz. The system structure i.e. increased viscosity negatively impacted the inactivation efficacy, most likely by reducing bubble collapse intensity and subsequent ultrasonic effects i.e. streaming.A systematic study of the effects of combined treatment order (nisin and ultrasound) and system structure in viscoelastic XG-based food model systems, with a comparison between E. coli was observed for the combination of nisin and ultrasound at 500 kHz, but only when nisin was applied before ultrasound treatment, despite nisin alone having no effect on Gram negative bacteria. Advanced microscopy techniques showed that on a cellular level for the combined treatment, total destruction of the cells occurred while ghost cells were present in systems treated with ultrasound only. Using a combination of kinetics, flow cytometry, microscopy and sonochemiluminescence, the mechanism of ultrasound inactivation was suggested to require a physical aspect resulting in terminal damage to the cell wall and membrane, and the subsequent efflux of cellular contents. Chemical effects are likely to also play a part. Ultrasound in combination with nisin is thought to enhance inactivation by allowing nisin to penetrate the cytoplasmic membrane, but only when nisin is present during sonication. Future work should aim to identify a condition or combination of treatments to which Listeria species are not resistant, and incorporate other species such as natural microflora, for a more advanced understanding of ultrasound/nisin efficacy and stress adaptation/resistance. Further optimisation of the ultrasound mode, and investigation of sonochemical versus sonomechanical effects, may also be necessary.Enhanced inactivation of Katherine M. Costello: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Writing – original draft, Writing – review & editing. Eirini Velliou: Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing – review & editing. Jorge Gutierrez-Merino: Conceptualization, Methodology, Project administration, Resources, Supervision, Writing – review & editing. Cindy Smet: Methodology, Supervision, Writing – review & editing. Hani El Kadri: Data curation, Investigation, Methodology, Writing – review & editing. Jan F. Van Impe: Funding acquisition, Supervision, Writing – review & editing. Madeleine Bussemaker: Conceptualization, Methodology, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.