Tea can be found among the most widely consumed beverages, but it is also highly susceptible to fraudulent practices of adulteration with other plants such as chicory to obtain an illicit economic gain. Simple, feasible and cheap analytical methods to assess tea authentication are therefore required. In the present contribution, a targeted HPLC-UV method for polyphenolic profiling, monitoring 17 polyphenolic and phenolic acids typically described in tea, was proposed to classify and authenticate tea samples versus chicory. For that purpose, the obtained HPLC-UV polyphenolic profiles (based on the peak areas at three different acquisition wavelengths) were employed as sample chemical descriptors for principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) studies. Overall, PLS-DA demonstrated good sample grouping and discrimination of chicory against any tea variety, but also among the five different tea varieties under study, with classification errors below 8% and 10.5% for calibration and cross-validation, respectively. In addition, the potential use of polyphenolic profiles as chemical descriptors to detect and quantify frauds was evaluated by studying the adulteration of each tea variety with chicory, as well as the adulteration of red tea extracts with oolong tea extracts. Excellent results were obtained in all cases, with calibration, cross-validation, and prediction errors below 2.0%, 4.2%, and 3.9%, respectively, when using chicory as an adulterant, clearly improving on previously reported results when using non-targeted HPLC-UV fingerprinting methodologies. Food integrity, quality and safety are of paramount importance to society. The selection of a specific food product is based on consumers’ benefit perception, which depends on a great variety of properties . Food orCamellia sinensis plant (an evergreen shrub native to East Asia and China). The characteristic aroma and flavor of tea infusions, and their numerous recognized health-beneficial attributes , are responsible for the high consumption of this drink on a daily basis [Camellia sinensis var. assamica), produced only in the region of Yunnan (China). This variety is processed in humid conditions to allow bacteria composting action. Finally, oolong tea is a controlled fermented variety where oxidation is limited to 10–70%.Tea is an aromatic drink obtained from the leaves of the ly basis ,7,8,9,10Chicorium intybus), a perennial herbaceous plant belonging to the Astareceae family, was studied as a tea adulterant as its use in tea must be completely banned when non-declared because of possible health effects [Tea is one of the most adulterated drinks, with cereal starch, legume husks and chicory as common adulterants of tea ,12. In t effects ,14.The classification and authentication of teas has been addressed either by non-targeted or targeted analysis, together with multivariate chemometric methodologies. For example, a targeted determination of metal elements by inductively coupled plasma-mass spectrometry (ICP-MS) followed by linear discriminant analysis was described to assess the region of production of several green teas from China . PolypheIn the present contribution, a simple HPLC-UV polyphenolic profiling method followed by chemometrics was developed to detect and quantify adulterated tea samples (using chicory as adulterant). With this in mind, seventeen phenolic compounds, typically described in tea samples, were selected. Samples were analyzed by C18 reversed-phase HPLC-UV, and peak areas of the identified compounds were used as chemical descriptors for chemometrics. A total of 120 samples including five tea varieties and chicory were analyzed for classification using principal components analysis (PCA) and PLS-DA. Then, adulteration cases based on the adulteration of each tea variety with chicory, as well as the adulteration of red tea with oolong tea, were processed by PLS regression to study the capability of the proposed HPLC-UV polyphenolic profiling method for the detection and quantitation of adulterant levels in tea extracts.Acetonitrile  from PanReac AppliChem , and formic acid (≥98%) from Sigma-Aldrich  were used. Purified water  was employed. A commercially available mineral water obtained from Eroski  was used for tea and chicory brewing.p-coumaric, 4-hydroxybenzoic and 3,4-dihydroxybenzoic acids, and quercetin, (−)-epicatechin, rutin, apigenin, and kaempferol were purchased from Sigma-Aldrich; (+)-catechin and myricetin from Fluka ; and (−)-epigallocatechin from Biosynth Carbosynth .Seventeen polyphenolic and phenolic acid chemicals were employed, all of them of analytical grade. Gallic, caffeic, ferulic, vanillic, sinapic, syringic, −1) were prepared in methanol or DMSO (depending on the compound). Working solutions were obtained by the corresponding dilution with Milli-Q water of the stock standards.Stock standard solutions of the targeted polyphenolic chemicals , equipped with a C18 guard-column , provided by Phenomenex  using a HPLC instrument from Agilent HPLC 1100 Series  equipped with a G1312A binary pump, a WPALS G1367A automatic sample injector, a G1315B diode-array detector, and a PC with the Agilent Chemstation software (2001–2012 version).Chromatographic separation was performed on a Kinetex−1 as mobile phase flow rate; 5 µL of injection volume; and room temperature for column separation. Ultraviolet-visible (UV-vis) acquisition was performed at 280, 325 and 370 nm, where the targeted polyphenolic compounds presented maximum UV-vis absorption spectrum values, and due to the fact that they resulted to be the most discriminant ones for classification purposes based on previous studies with the kind of samples analyzed.A gradient elution separation was proposed for the determination of polyphenols and phenolic acids, as well as other components of the analyzed samples; solvent A was 0.1% formic acid in water and solvent B was acetonitrile. Elution program: 0–1 min from 5–10% solvent B; 1–4 min from 10–16% solvent B; 4–8 min, isocratic at 16% solvent B; 8–8.5 min, from 16–25% solvent B; 8.5–13.5 min, from 25–60% solvent B; 13.5–16 min, from 60–100% solvent B; 16–16.5 min, isocratic at 100% solvent B; 16.5–16.6 min, back to initial conditions at 5% solvent B; and 16.6–22 min isocratic at 5% solvent B for column re-equilibration. Other conditions: 800 µL minA total of 100 teas of different varieties including white, black, green, red and oolong extracts (20 samples/variety), as well as 20 chicory extracts, obtained from different markets in Barcelona (Spain), were employed. The description of the samples  and the number of lots employed for each sample is provided in Tea and chicory extracts were obtained following a previously established procedure ,23. BrieSamples were randomly analyzed with the HPLC-UV methodology. In addition, a QC, a polyphenolic standard mixture, and a blank  were analyzed after every 10 samples. Polyphenolic and phenolic acid peak areas detected at the three registered acquisition wavelengths were then measured and used to generate the profiling data matrices, which were then submitted to PCA, PLS-DA, and PLS regression methods using SOLO 8.6 chemometric software from Eigenvector Research . The theoretical background of these methods is addressed in reference . For allTo validate the obtained PLS-DA models, 60% of the samples (randomly selected) were employed for calibration while the other 40% were used for prediction. To evaluate the predictive performance of the classification models, the sensitivity was calculated as TP/(TP + FN), the specificity as TN/(TN + FP), and the accuracy as (TP + TN)/TS, all of them expressed as a percentage, with TP being the number of positive samples correctly assigned to the class, TN the number of negative samples correctly assigned , FN the number of false negatives incorrectly assigned as not belonging to the class, FP the number of false positives incorrectly assigned to the class, and TS the total number of samples.Adulteration cases studied by PLS consisted of each one of the five different tea samples adulterated with chicory, as well as red tea adulterated with oolong tea. For that purpose, the PLS calibration set comprises adulteration levels of 0, 20, 40, 60, 80 and 100%, whereas adulteration levels of 15, 25, 50, 75 and 85% were used for validation. Each one of the adulteration levels employed was done by quintuplicate. For the preparation of these adulterations, five pooled tea samples were prepared for each tea variety by mixing 10 different samples (different origins) of the same variety, and each pooled tea sample was then adulterated with a different chicory sample. Thus, the five replicates for each adulteration level were obtained using different combinations of tea/chicory samples. In previous publications, we evaluated the suitability of non-targeted fingerprinting strategies based on HPLC-UV and HPLC-FLD  and FIA-® C18 porous-shell reversed-phase column using gradient elution with acetonitrile and 0.1% formic acid as mobile phase components (flow rate 800 µL min−1) was employed , as a compromise between analysis time and peak resolution, the obtained chromatographic separation was considered acceptable for the intended purpose of this work. It should be taken into account that despite the presence of some polyphenolic coelutions, the aim of the present work is not the quantification of phenolic compounds but the monitoring of their peak signals as potential descriptors to accomplish the classification and authentication of the analyzed samples. In those coelution cases where the signal of both compounds cannot be distinguished based on their different response at the three-acquisition wavelength, it must be considered that the signal peak area employed for further studies will include the contribution of both coeluting compounds.A total of 17 flavonoids and phenolic acids typically occurring in tea samples were selected as the targeted compounds . As repoTea and chicory samples were extracted as described in The differences observed in the chromatograms, not only on the targeted polyphenolic compounds detected but also on their corresponding peak signals, and the fact that the obtained HPLC-UV polyphenolic profiles seem to be reproducible for all the samples within the same class variety, suggested that the targeted HPLC-UV polyphenolic profiling data could be proposed as sample chemical descriptors to study the classification of tea and chicory by chemometrics.The signal peak areas of the 17 targeted flavonoid and phenolic acid compounds at the three monitored acquisition wavelengths in all the analyzed tea and chicory samples were employed to build the X-data matrix for chemometrics. The resulting data matrix had a dimension of 133 × 30 (tea/chicory/QC samples × detected polyphenolic peak areas at three wavelengths). It should be commented that some compounds are not detected at some of the registered acquisition wavelengths. This matrix was first subjected to PCA to explore the sample distribution and evaluate data repeatability, as well as the robustness from the QCs behavior.The score plot of PC1 versus PC2 is depicted in Focusing on the analyzed samples, they are grouped based on their variety. Chicory samples appeared at the left-bottom area of the plot , close to red tea samples. This last group, together with the oolong samples, are grouped compactly, and mainly presenting negative values for PC1 and PC2. In contrast, white and black tea samples are mainly characterized as presenting positive PC2 values, while green tea samples have positive PC1 values, appearing mainly in the right area of the plot. In any case, chicory samples are clearly discriminated from all the tea varieties . A noteworthy result is that acceptable sample discrimination was also observed with the five tea varieties when using the polyphenolic profiles based on the peak areas of seventeen polyphenols at three different acquisition wavelengths. This good sample discrimination was not observed in previous studies when either non-targeted HPLC-UV-FLD or FIA-MS fingerprints were employed ,23, wherThe X-data matrix see  without A paired PLS-DA model was built to classify the tea varieties analyzed versus chicory. With this in mind, 60% of the samples randomly selected were used for calibration, while the remaining 40% (considered “unknown samples”) were used for validation and prediction. p-coumaric acid, and apigenin, among others, are discriminant of green tea. In the case of chicory samples, caffeic acid could be proposed as a discriminant polyphenolic.The loading plot b shows tThe good results obtained in the classificatory study demonstrate the good performance of the polyphenolic profiles (by monitoring only 17 flavonoids and phenolic acids at three acquisition wavelengths) for the classification of tea and chicory samples.2 values higher than 0.996) were obtained, with calibration, cross-validation and prediction errors below 2.0%, 4.2% and 3.9%, respectively. This is better than those previously reported by using non-targeted HPLC-UV or HPLC-FLD fingerprints [Sample chemical descriptors based on the obtained polyphenolic profiles were also used for the detection of tea frauds and the quantitation of tea adulteration levels with chicory by PLS. For that purpose, the five possible adulteration cases, i.e., each tea variety adulterated with chicory, were addressed. Adulteration levels for both calibration and validation in PLS are described in erprints , and eveerprints , demonst2 0.995), and calibration, cross-validation and prediction errors of 2.3%, 3.9% and 5.9%, respectively. These results confirm that the evaluated targeted HPLC-UV polyphenolic profiles also work as good chemical descriptors to authenticate teas when different tea varieties are blended.As a proof of concept, the authentication of tea samples blended with teas of other varieties was also evaluated. For that purpose, the adulteration case based on a red tea extract blended with an oolong tea extract (considered as the adulterant), was also studied, and the obtained PCA and PLS data is provided in In summary, the obtained results in this study confirm that HPLC-UV polyphenolic profiles are feasible and good chemical descriptors to characterize, classify and authenticate tea samples. In addition, data is useful to detect and quantify the levels of chicory in tea adulteration issues and to assess the authentication of teas when different varieties are blended.


Inoculation strategy is a significant determinant of the flavor quality of Zhenjiang aromatic vinegar. Herein, the comparative analyses of the effects of various inoculation strategies on the physicochemical properties, microbial community structure, and flavoring characteristics of Zhenjiang aromatic vinegar were performed. The results showed that the contents of total acid (6.91 g/100 g), organic acid (2099.63 ± 4.13 mg/100 g) and amino acid (3666.18 ± 14.40 mg/100 g) in the direct inoculation strategy were higher than those in the traditional inoculation strategy . At the same time, it can effectively promote the production of acetoin. The diversity of strains under the traditional inoculation strategy was higher than that under the direct inoculation strategy, and the relative abundance of major microbial genera in the fermentation process was lower than that under the direct inoculation strategy. In addition, for two different inoculation strategies, pH was proved to be an important environmental factor affecting the microbial community structure during acetic acid fermentation. The correlation between main microbial species, organic acids, non-volatile acids, and volatile flavor compounds is more consistent. Therefore, this study may help to develop direct injection composite microbial inoculants to replace traditional starter cultures in future research. Due to Because the quality of vinegar produced by traditional inoculation is mostly adjusted by experience, the metabolic mechanism and environmental parameters of complex fermentation microbial population cannot be controlled. This leads to low output and unstable quality of products, which seriously hinders technological progress and expansion of production scale. Therefore, the study of the relationship between microbiota and the formation of flavoring substances is important for understanding, controlling and effectively improving vinegar fermentation to improve food quality, productivity and safety. The rapid development of molecular biology and modern detection technology has gradually revealed the contribution of core microorganisms and functional microflora to the flavor of fermented products. In addition, breeding excellent functional strains and adjusting microbial community function through direct inoculation to improve fermentation efficiency are one of the most effective measures to improve yield and quality. Compared with traditional inoculation strategies, microbial community with selective species proportion is simplified and fermentation is easier to control .Aspergillus niger-enhanced gluten meal in the production of Baoning Vinegar can increase the total acid and organic acid contents as well as the utilization rate of starch and maintain relatively stable quality throughout the three cycles of fermentation  to replace the traditional fermented and mature seed grains in fermentation production. Under the same conditions, compared with the traditional inoculation strategy, the direct injection inoculation strategy can strengthen the adaptability and stability of the functional bacteria directly inoculated in the fermentation system, and effectively improve the product quality. Moreover, it can be applied in food and pharmaceutical fields . Relevanentation . Lactoba acetoin . Additiotabolism . TherefoTherefore, acetic acid and lactic acid bacteria, the key microorganisms in the production of Zhenjiang vinegar, were introduced into the solid-state fermentation production as direct inoculants. The effects of two different inoculation strategies on the physicochemical properties , microbial community structure and flavor characteristics  of Zhenjiang vinegar were compared. In addition, the effects of two inoculation strategies on microbial communities and metabolic components, and their differences in contribution to vinegar flavor were studied. The purpose of this study was to explore the effects of two inoculation strategies on the physicochemical indexes of Zhenjiang vinegar during acetic acid fermentation, and to provide theoretical support for the combination of traditional and modern vinegar brewing methods and the adjustment of technological parameters in large-scale production.2.2.1.Acetobacter A-1 and Lactic Acid Bacteria Y-14) of Zhenjiang aromatic vinegar were used as research subjects. The marinade samples collected on days 0, 2, 4, 6, 8, 10, 12, 14, 16, and 18 of the fermentation were sampled daily before the rotation of the fermented grains, with three replicates for each sample. The vinegar was extracted from the fermentation tank from top to bottom, and 100 g of it was weighed in a self-sealing bag after uniform mixing. A 30-mL sample of marinade was transferred from the bottom of the cylinder to a 50-mL centrifuge tube and stored at −20°C. Samples were thawed in a water bath at room temperature (26°C to 30°C) before analysis.In this study, Zhenjiang aromatic vinegar was brewed by traditional solid-state fermentation process. The natural seed fermented grains inoculated on the seventh day of fermentation  and artificially pure cultured strains . Citric acid (≥99.5% purity), L-pyroglutamic acid (≥99% purity), lactic acid (≥99% purity), acetic acid (≥99% purity), succinic acid (≥99% purity), and malic acid (≥99% purity) were purchased from McLean Biochemical Technology Co., Ltd .2.3.pei was added into 30 ml distilled water, agitated at 100 rpm for 3 h at room temperature (20°C), centrifuged for 10 min at 6000 g, and the supernatant collected for the following analysis. The total acid, pH, amino nitrogen, and reducing sugar contents were determined following the previous methods  under the condition of 97% similarity. Moreover, the most abundant sequence (MOST ABUNDANT) was selected as the OTU representative sequence. Using SILVA 132 as the reference database, the naive Bayesian classifier was combined with the alignment method for taxonomic annotation of the sequences, with a 70% confidence threshold. Sample α diversity index was evaluated using the Mothur V1.30 software. Furthermore, β diversity was analyzed with QIIME software, and the similarity of species diversity among various samples was compared.2.5.3.p value, which were analyzed and plotted using GraphPad Prism 7.0 and R language and other software.Spearman correlation analysis was performed on the relative abundance of >1% and the difference or key strain with the physicochemical factors and main volatile flavor substances in vinegar-fermented grains, respectively, to calculate the correlation coefficient R-value and significant difference 3.3.1.To study the dynamic changes in physicochemical parameters and flavoring substances during acetic acid fermentation of Zhenjiang aromatic vinegar under different inoculation strategies, the total acid , reducinvia direct inoculation was 6.91 g/100 g.The total acid content of vinegar-fermented grains exhibited a fluctuating upward trend throughout the acetic acid fermentation process, which was a key indicator of vinegar acidity. On days 0–7 of acetic acid fermentation, the total acid content continued to increase owing to the mass propagation of microorganisms such as lactic acid bacteria. Aerobic microorganisms such as acetic acid bacteria proliferate in a large number and generate a large amount of organic acids including acetic acid and lactic acid for 7 to 18 days after the fermented grains are completely turned over for fermentation in order that the total acid content increases rapidly . At the pei, the macromolecular sugar contained in raw material bran is hydrolyzed into small molecular sugar by acid, such as monosaccharide and other reducing sugar. At the same time, the temperature reached the optimum temperature of saccharifying enzyme (35°C-40°C), the activity of the enzyme increased, and the reducing sugar produced by hydrolysis of raw materials was accumulated. Because the activity and acid-producing ability of acetic acid bacteria under the direct inoculation strategy is stronger than that of the traditional inoculation strategy, it can fully acidolysis macromolecular sugar and produce more reducing sugar. In the late stage of acetic acid fermentation, microorganisms proliferate, nutrients are rapidly consumed, and reducing sugar content is significantly reduced.The sweet flavor of Zhenjiang aromatic vinegar is primarily a result of the saccharification of food. These saccharides can enhance the flavor of vinegar. Simultaneously, the reducing sugar of vinegar can react with amino acids to produce flavor substances . On the The non-volatile acids can neutralize the strong acidity of acetic acid and make the finished vinegar taste soft and delicate, which is the key to the quality of Zhenjiang balsamic vinegar . Lactic Pei under the direct inoculation strategy continued to rise until the fermentation temperature reached 48.0°C on the twelfth day and then began to decrease to 35.33°C ± 0.35°C. It was possible that the direct inoculation of acetic acid bacteria contained a high concentration of lactic acid bacteria and produced more acid and heat, which inhibited the growth of other bacteria.Temperature affects the metabolic activity of microorganisms and the changes of alcohol and water during the fermentation process, making it a comprehensive indicator used to determine the fermentation quality of vinegar-fermented grains. Under optimal conditions, the rapid propagation and metabolism of microorganisms not only rapidly converted the alcohol in vinegar-fermented grains into acetic acid but also metabolized a large number of organic acids, amino acids, esters and other substances. The fermentation temperature ranged from 38°C to 46°C, which was the most active phase of acetic acid bacterial metabolism. A large number of products were accumulated, and the primary flavoring substances of aromatic vinegar were generated, alongside a large amount of heat. During the final phase of fermentation, owing to the consumption of nutrients and large accumulation of metabolites, the metabolism of microorganisms such as acetic acid bacteria was inhibited, and the temperature was reduced and maintained at 35°C . Using tOverall, there were considerable differences in total acid, nonvolatile acid, reducing sugar, the temperature of fermented vinegar, organic acid, and organic acid content between the two inoculation strategies . Moreovevia fermentation and reached 1939.66 ± 4.16 mg/100 g dry fermented grains at the end of AAF, among which acetic acid, lactic acid, and citric acid were the highest produced. Using the direct inoculation strategy, the organic acid content increased to 2099.63 ± 4.13 mg/100 g dry fermented grains on the 14th day of AAF and then decreased slightly, with acetic acid, citric acid, and tartaric acid accounting for most , nut and baking aroma). According to reports, TTMP is the primary bioactive component in vinegar . As fermEsters are the most abundant volatile flavoring substances, and ethyl acetate and ethyl lactate had the highest concentrations in the entire process, which was directly correlated with the higher concentrations of the synthetic precursors of acetic acid and lactic acid . The env3.2.The structure and succession pattern of bacterial and fungal communities throughout the entire fermentation process for the two inoculation strategies was investigated by using amplicon sequencing analysis. Significant differences were found between the Chao1 and Shannon indices of acetic acid fermentation bacteria and fungi for the direct inoculation strategy and those for the traditional inoculation strategy . The ChaThroughout the entire process of acetic acid fermentation, bacteria predominate the metabolic activities in vinegar culture, and their biomass in vinegar-fermented grains is higher than that of fungi . Fungi aAcetobacter continue to increase. During this stage, the predominant microorganisms were lactic acid bacteria and Acetobacter, including Komagataeibacter and Gluconacetobacter. In addition to oxidizing alcohol substrates such as ethanol and sugar alcohols via the respiratory chain to organic acids such as acetic acid and lactic acid, Acetobacter could promote the production of certain ketone and aldehyde flavoring substances  provide selective strength for the ‘systematic self-domestication’ of microbial communities, thus shaping the decision-making process of microbial succession .Environmental factors had a significant impact on the fermentation of vinegar in the solid state. In addition to the indoor temperature, humidity, and other external environments of fermented grains, the total acid, nonvolatile acid, and reducing sugar of vinegar-fermented grains were not only metabolites of acetic acid fermentation but also substrates of vinegar production, making them crucial environmental variables in vinegar-fermented grains. Redundancy analysis (RDA) and Spearman were used to conduct correlation analysis on seven bacterial and seven fungal groups (with average relative abundance >10%) and four environmental parameters to determine the relationship between the succession of microbial communities during the acetic acid fermentation stage and environmental changes caused by different inoculation strategies .Acetobacter pasteurianus, Lactobacillus acetotolerans, and Saccharomyces sp (unclassified) were significantly positively correlated with reducing sugar, nonvolatile acid, and total acid. Acetobacter pasteurinus has strong ethanol resistance and can convert ethanol in fermented grains into lactic acid, acetic acid and other substances , and its relative abundance will increase with acetic acid fermentation  were significantly negatively correlated with pH. Xanthomonas, Lactobacillus and Alternaria (unclassified) were significantly positively correlated with the pH value of the direct inoculation strategy. Therefore, under different inoculation conditions, pH has the most significant effect on microorganisms. The pH value will affect the activity of microorganisms such as Acetobacter and Lactobacillus during acetic acid fermentation. When the pH value in the fermentation broth is too high or too low, the growth of microorganisms will be inhibited. In addition, pH value can also affect the accumulation of nutrients in the fermentation broth and the stability of the metabolic substances of acetic acid bacteria, thus reducing the acid production capacity of acetic acid bacteria  and flavoring substances formed during the fermentation process, The red line represents positive correlation, and the blue line represents negative correlation ,B, 6A,B. acetate , 6A. Whimic acid , 6B. Thike notes .Acetobacter and Lactobacillus, other major microorganisms, such as Agrobacterium Larrymorei under the traditional inoculation strategy, have a positive correlation with tartaric acid Agrobacterium Larrymoorei was positively correlated with tartartaric acid, and negatively correlated with pyroglutamic acid, lactic acid and pyruvic acid. Stenotrophomonas Maltophilia was negatively correlated with pyroglutamic acid and pyruvic acid. Xanthomonas Sacchari was positively correlated with tartartaric acid and negatively correlated with acid. Agrobacterium larrymoorei, Stenotrophomonas maltophilia, Xanthomonas Sacchari were positively correlated with 3mehis, dimethyl ether and ethane, 1,1-diethoxy-and negatively correlated with lAsn, Thr, Ala, a-ABA, Asp., Ile, Cys, Arg, P-ser and Acetoin. Agrobacterium larrymoorei, Stenotrophomonas maltophilia, Xanthomonas Sacchari under the direct inoculation strategy, were positively correlated with 11 alcohols, 22 esters, 6 aldehydes and 3 acids, and negatively correlated with 6 alcohols, 7 esters, 1 aldehyde and 7 acids, and these strains are negatively correlated with ethyl 4-acetoxybutyloate, Butanoic acid, 3-methyl-, 2-methody-4-vinylphenol, and with hexadecanoic acid, ethyl ester, pexanoic acid, ethyl ester, octanoic acid, ethyl ester, benzoic acid, ethyl ester, heptanoid acid, ethyl ester, 1-Nonal, Pentanoic acid, ethyl ester, Nona Noic acid, ethyl ester and diethyl azelate are positively correlated.In addition to the most important Saccharomyces Unclassified, the main fungus under the traditional inoculation strategy is positively correlated with acetic acid, pyruvic acid, Asn, Thr, Ala, a-ABA, Asp., Ile, Cys, Arg, P-ser, Gly, Lys, PEA, and negatively correlated with tartaric acid. While, Saccharomyces Unclassified, the main fungus under direct inoculation strategy is positively correlated with both malic acid and acetic acid. Saccharomyces cerevisiae strains with strong fermentation capacity and strong fragrance production capacity for pure and mixed fermentation, and found that the content of volatile flavor substances could be increased when two excellent yeast strains were mixed for fermentation  under different inoculation strategies in the fermentation process, it was found that pH had the most significant effect on microorganisms under different inoculation strategies.Finally, the Spearman correlation coefficient was used to study the correlation between important microorganisms (average relative abundance>10%) and flavoring substances formed during fermentation under different inoculation strategies. It was found that in the direct inoculation strategy, the correlation between major microbial species and organic acids, non-volatile acids and volatile flavor compounds was more consistent.The results revealed the effects of different inoculation strategies on the microbial composition and flavor quality during the fermentation of Zhenjiang aromatic vinegar, which will provide useful information for the future development of a direct-injection composite microbial inoculum to replace the traditional starter.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/XY conducted experiments and made significant contributions to the acquisition, analysis, and interpretation of data. YZ and JL participated in the experiment and revised and discussed the manuscript. KW and PL participated in the revision of the manuscript. YY participated in the experiment and reviewed and agreed to publish this article. ZY and YW designed the study and participated in the drafting and revision of the manuscript. All authors contributed to the article and approved the submitted version.This project was supported by the Postgraduate Research & Practice Innovation Program of Jiangsu Province (No. 1182162204) and the National Natural Science Foundation of China (No. 32072202).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The histopathological, histomorphometric, and immunohistochemical analyses gave comparable results for these bone substitute materials in terms of newly formed bone: , residual biomaterial , and soft tissue , with no significant difference found between the groups , proving that BCP is equally suitable and successful for alveolar bone regeneration.Following trauma, chronic periapical process, or tooth extraction, a large loss of bone volume is noticed during the healing process. To facilitate the placement of dental implants, various surgical procedures are used for an optimal alveolar ridge profile, while maintaining adequate bone dimensions. The main aim of this study was to determine the healing ability  of alveolar bone defects during augmentation with two different biomaterials: injectable biphasic calcium phosphate (BCP) and anorganic bovine bone (ABB). Thirty-eight subjects were randomly divided into two groups. The first group received the tested bone substitute biomaterial (BSB), i.e., BCP (maxresorb inject Following trauma, chronic periapical process, or tooth extraction, a large loss of bone volume is noticed during the healing process ,2. Two-tXenografts are used as an alternative to the gold standard. Most scientific research mentions deproteinized bovine bone, porcine bone, and more recently, horse bone . DeproteThe term alloplastic bone grafts refers to synthetic biomaterials. Synthetic materials currently exhibit only osteoconductive properties. Materials that belong in this category include metals, polymers, polyglycolides, calcium phosphate cements, HA, tricalcium phosphate (TCP), and bioglass ,44. The Also, after tooth extraction, the supply of metabolic products is interrupted. Immune cells produce various cytokines that are temporally and spatially controlled at the site of injury and cause acute inflammation, angiogenesis, and accumulation of mesenchymal cells. The recruited osteoprogenitor cells produce bone morphogenetic protein 2 (BMP-2), which, in coordination with other factors, promotes local accumulation and osteogenic differentiation of mesenchymal cells at the site of injury ,54,55,56The main aim of this study was to determine the healing ability  of alveolar bone defects during augmentation with two different biomaterials: injectable biphasic calcium phosphate (BCP) and anorganic bovine bone (ABB). The ability of BSB to restore damaged tissue was quantitatively and qualitatively evaluated, and pathohistological changes were described on bone biopsies six months after augmentation. In addition, the expression of Osx and BMP-2 in bone remodeling and mesenchymal cell differentiation was detected by immunohistochemistry.The study included 38 participants, of whom 17 were males (45%) and 21 were females (55%). They were divided into two groups: the test group and the control group. The mean age of the respondents was 35.03 ± 9.16 years . A brief description of the characteristics of the sample itself can be found in In the quantitative analysis, the areas of newly formed bone, residual BSB, and soft tissue structures were determined with respect to the total area of the histological sample in the field of view. After processing all the results, the obtained areas were converted into volume percentages (%), as shown in t-test was used to compare the small samples. The t-test was used to compare each variable separately, i.e., newly formed bone (t (38) = 0.487; p = 0.629), residual biomaterial (t (38) = 0.705; p = 0.485), and soft tissue (t (38) = −1.626; p = 0.113), as shown in Since the requirements for performing parametric tests were met, the t-test values, there is no statistically significant difference between the test group and the control group in terms of newly formed bone, residual biomaterial, and soft tissue.As shown by the In the qualitative analysis, the pathohistological response of the host tissue to the used BSB was evaluated, i.e., osteoblasts, osteocytes, fibroblasts, fibrocytes, blood vessels, and cells of the monocyte–macrophage system were described. In addition, the newly formed bone, the residual biomaterial, and the soft tissue were described.Representative samples of the control group and histological staining with HE under magnification of 100, 200, and 400 times and labelling of newly formed bone, residual biomaterial, soft tissue, osteoblasts at the boundary between newly formed bone and residual biomaterial, osteocytes in newly formed bone and Howship lacunae in the bone bed with a blood vessel indicating the integration of biomaterials and newly formed bone . Representative samples of the test group and histological staining with HE under magnification of 100, 200, and 400 times and labeling of newly formed bone, residual biomaterial, soft tissue, osteoblasts at the boundary between newly formed bone and residual biomaterial, osteocytes in newly formed bone and Howship lacune in the bone bed with a blood vessel indicating the integration of biomaterials and newly formed bone .Representative samples of the control group and histological staining with HE under magnification of 100, 200, and 400 times and labeled fibroblasts and fibrocytes in the soft tissue and cells of the monocyte–macrophage system around the residual biomaterial, indicating biomaterial degradation .Representative samples of the test group and histological staining with HE under magnification of 100, 200, and 400 times and labelled fibroblasts and fibrocytes in the soft tissue and cells of the monocyte–macrophage system around the residual biomaterial, indicating the decomposition of the biomaterial .Immunohistochemical analysis of bone biopsy samples was performed to detect Osx transcription factor and BMP-2 protein.Representative samples of control group and Osx immunohistochemical staining at magnification of 100, 200, and 400 times  showed nRepresentative samples of the test group and immunohistochemical Osx staining under magnification of 100, 200, and 400 times  and labeRepresentative samples of control group and BMP-2 immunohistochemical staining under magnification of 100, 200, and 400 times  and labeRepresentative samples of the test group and BMP-2 immunohistochemical staining under magnification of 100, 200, and 400 times  and labeThis study included 38 subjects, 17 (45%) male and 21 (55%) female. They were divided into two groups, the test group and the control group. The mean age of the respondents was 35.03 ± 9.16 years . Parallels can be drawn with human studies conducted by Čandrlić et al. , Jelušić®) was used, were expressed as mean ± standard deviation of the mean and consisted of the following: newly formed bone (41.73 ± 13.99%), residual biomaterial (31.72 ± 15.52%), and soft tissue structures (26.54 ± 7.25%). On the other hand, the pathohistological response of the host tissue to the used BSB was evaluated by qualitative analysis.Quantitative and qualitative analysis of bone biopsy samples is used to evaluate regenerated augmentation areas, which is supported by many previous studies in addition to ours ,67,68. T® is one of the best-documented bovine xenografts and has been used as a control group in many studies [®); the effect of this was investigated in the control group in this study. This is the case with studies such as that of McAllister et al. [In the literature, Bio-Oss studies ,69,70. F studies  for the r et al. ,73: In tr et al. ,75 descrr et al. , the per® in terms of newly formed bone with 39 ± 1.6%, 31 ± 1.4%, residual BSB, and 34 ± 1.6% soft tissue structures, which is the closest to the results obtained in this study six months after augmentation with the same BSB. It is interesting to mention the recent study by Wei et al. [® in socket preservation after molar extraction in patients with periodontitis. Although such patients were excluded in our study, the study by Wei. et al. showed promising results in terms of an increase in alveolar bone dimensions in the maxilla and mandible 6 months after augmentation. These conclusions confirm a good regenerative potential of Bio-Oss® in alveolar bone regeneration as shown in our study. Conversely, some human studies have shown that alveolar ridge defects grafted with Bio-Oss® resulted in different percentages of newly formed bone, residual BSB, and soft tissue structures from the above results, such as the studies by Froum et al. [According to the literature on human studies using this form of xenograft six months after augmentation, an older study by Zitzmann et al. in 2001  confirmei et al.  investigm et al. , Schmittm et al. , Lorenz m et al. , Fienitzm et al. , etc. Mom et al. , Pignatom et al. , and Sanm et al. . Such re® show good integration between the biomaterial and the surrounding host tissue [®—31.72 ± 15.52%) than in the test group (maxresorb inject®—28.61 ± 11.38%) and that xenograft resorption was ultimately slower. Bio-Oss® has proven to be a valuable alternative to the gold standard from a clinical point of view, ensuring good quality of the newly formed bone and promising a long-term regeneration rate [In addition to histomorphometric analysis, the interest of previous studies, such as this one, focused on the interactions between BSB and host tissues, such as the biological response of the tissue related to the origin of BSB, which is crucial for qualitative analysis. Notwithstanding the slow resorption demonstrated in many studies, the qualitative light microscopic analyses in other studies as well as this study strongly demonstrate that the sites treated with Bio-Osst tissue ,88. Furtt tissue ,89. Accot tissue . These fion rate .® inject) with a composition of 16.5% biphasic granules and 83.5% nano-HA gel, representative of the group of alloplastic BSB, was used in the test group. The quantitative results of this study, i.e., the comparison of newly formed bone, residual BSB, and soft tissue structures in the test group in which injectable BCP (maxresorb® inject) was used, are also given as mean ± standard deviation of the mean and were as follows: newly formed bone (39.91 ± 8.49%), residual biomaterial (28.61 ± 11.38%), and soft tissue structures (31.49 ± 11.09%).Injectable BCP  were reviewed and evaluated semiquantitatively. Intensity level 3 (+++), i.e., strong staining indicating good osteoconductive properties of both BSBs, was detected in both groups. Representative samples from the control and test group and Osx immunohistochemical staining showed cells with Osx expression of intensity level 3 (+++) in pre-osteoblasts anchored to the margin of newly formed bone, indicating their transition into mature osteoblasts and osteocytes. In addition, trabecularization of the new bone was observed, indicating continuous remodeling. Osx has been shown to be involved in osteoblast differentiation, maturation, and activity; it regulates the expression of various markers, i.e., osteoblast proteins, the most important of which are osteopontin (OPN) and osteocalcin (OCN), etc., as indicated by the currently available literature. Previous studies have shown that Osx is an essential transcription factor in osteogenic differentiation ,106.On the other hand, representative samples from the control and test groups and BMP-2 immunohistochemical staining showed cells with BMP-2 expression levels of 3 (+++), present mainly in the zones where differentiation of mesenchymal cells into pre-osteoblasts continued, indicating recovery, i.e., regeneration of the damaged tissue. According to the literature, BMP-2 is a protein that acts as a potent osteogenic factor and promoter of osteoblast differentiation, which was expressed in similar studies with this type of BSB, confirming the results obtained ,107.Thirty-eight subjects participated in this study. The main criteria for enrollment were that the patients had at least one tooth scheduled for extraction, all previous therapeutic options had been exhausted, and they had the possibility of dental implant placement at the extraction site after alveolar ridge augmentation. The criteria for inclusion were (1) age of subjects ≥18 and ≤60 years, (2) understanding of the protocol and informed consent signed by each subject, and (3) satisfactory physical and mental health of the subjects. Subjects who had some of the exclusion criteria were excluded from the study: (1) at least one absolute contraindication to implant therapy described by Wang and Hwang 2006. ; (2) sub®, 3M Deutschland GmbH, Neuss, Germany) was administered. Patients who agreed to participate in the study were randomized into two groups. The first group consisted of patients who received the test BSB , and the second group consisted of patients who received an alternative to the gold standard, ABB . The socket was filled with injectable BCP in the test group and with ABB in the control group. Finally, after filling the defect, an absorbable collagen membrane  was fitted to the surgical wound in both groups and the mucoperiosteal flap was closed with nonabsorbable 5.0 monofilament suture . The surgical procedure described is shown in ® 220, Saronno, Italy) for postoperative care of the oral cavity. One week after augmentation, the patient was referred for a radiological examination of the augmented area by cone beam computed tomography (CBCT) to verify the stability of the BSB at the augmentation site. Before completion of the six months of healing, thepatient was invited to obtain a control CBCT scan, which measured the dimensions of the alveolar ridge at the site of implantation. Immediately thereafter, the second phase of the study was scheduled, i.e., biopsy of the alveolar bone with final placement of a dental implant.Before the start of the procedure, radiographs were taken at the planned tooth ex-traction sites. Patients who met the inclusion criteria were prescribed an antibiotic  one hour before the procedure; local anesthesia with 4% articaine and epinephrine 1:100,000  used to take the bone biopsy sample had a smaller inner diameter (2.5 mm) than the final drill from the standardized set for shaping and bed preparation of the dental implant . From an ethical point of view, this avoids excessive removal of healthy bone. The bone biopsy samples were then left in a 4% formaldehyde solution  and sent to the laboratory for histological and immunohistochemical analysis.®, Sigma-Aldrich, St. Louis, MO, USA) for two weeks, embedding in paraffin blocks, and sectioning. Six contiguous sections with a thickness of 5 μm were prepared with a microtome  and placed on slides after drying. Before staining, the tissue had to be rehydrated by placing it twice in xylene  for 15 min each, then in a descending series of alcohol , and finally in distilled water. The tissue was then stained with hemalaun-eosin (HE) using histological staining kits. Finally, dehydration was repeated in an increasing series of alcohols . Digital photomicrographs were taken using a light microscope  with an attached video camera  at magnification of 10, 20, and 40 times. In a qualitative analysis, the pathohistological response of the host tissue to the BSB used was evaluated, i.e., osteoblasts, osteocytes, fibroblasts, fibrocytes, blood vessels, and cells of the monocyte-macrophage system were described.A standardized protocol for histological preparation of mineralized bone samples was applied to the samples, which included the following: fixation in a 4% formaldehyde solution , dehydration in increasing alcohol concentrations , decalcification with ethyldiaminetetraacetic acid . All the photomicrographs were taken under the same conditions . Before starting the analysis, parameters such as setting the scale on the basis of the known distance and converting it to the unit of length (μm) were set. Also, the possibility of manual correction was set to exclude from the analysis any edge regions that were not completely clear or that represented an artifact. Each sample was analyzed individually such that the digital micrograph was adjusted using the threshold option, with manual manipulation allowing areas of interest (ROI) to be marked in different colors. Accordingly, the areas of newly formed bone, residual BSB, and soft tissue structures were determined in relation to the total area of the histological sample in the field of view, and after all the results were processed, the obtained areas were converted into volume percentages (%). All the preparations were additionally examined by two independent researchers.2O2 , followed by a 10-min wash in PBS. According to the manufacturer’s recommendations, the samples were incubated overnight at 4 °C with a rabbit polyclonal antibody to Sp7/Osx  and a rabbit polyclonal antibody to BMP-2 . Immunohistochemical analysis was performed on four histological bone biopsy samples to detect Osx transcription factor and BMP-2 protein. Bone tissue samples with a thickness of 5 μm were deparaffinized in xylene  and then rehydrated in alcohol of decreasing concentration . The next step was protein renaturation, which was achieved by incubating the slices in citrate buffer  in a water bath at 90° for 10 min. After cooling, the samples were washed in a physiological solution buffered with phosphate-buffered saline (PBS) at pH 7.2. Blockade of endogenous peroxidase activity to avoid nonspecific binding was performed with 0.3% hydrogen peroxide Ht-test was used to evaluate the significance of the difference between the two samples in the context of a normal distribution. Any p value less than 0.05 was considered significant.For statistical analysis, IBM SPSS  was used. The results of the Shapiro–Wilk test were used to determine whether the distribution was normal. The mean and standard deviation of the mean were used for all results. A ® inject might be as suitable and successful as BSB for alveolar ridge augmenatation. It is necessary to highlight the strengths of this study. The study was conducted as a randomized controlled human clinical trial comparing quantitative, qualitative, and immunohistochemical analysis of two BSBs. The histomorphometric, histological, and immunohistochemical analysis and the use of a standardized free program with a detailed description of the samples allowed the methodology to be reproduced and the results obtained to be compared with future studies. All the subjects in the study underwent a standardized protocol in terms of surgical procedures and time from bone augmentation to biopsy. However, due to the small number of samples processed, especially for immunohistochemical analysis, further clinical studies with a larger sample and a longer follow-up period could be used to draw definitive conclusions in this regard. Notwithstanding some methodological limitations, the comparable results obtained with injectable BCP compared to the gold standard alternative, i.e., ABB, represent a promising outcome for the purpose of alveolar ridge augmentation after tooth extraction and dental implant placement.Histopathologic, histomorphometric, and immunohistochemical analyzes of these two BSBs showed comparable results and proved that maxresorb


Sudden cardiac death (SCD) is the leading cause of mortality globally (violent or non-violent), with few to no feasible remedies. The etiopathogenesis of SCD involves a complex and multilayered substrate in which dynamic factors interact with a preexistent cardiovascular pathology, which is often undiagnosed and untreated, leading to the rapid development of cardiac rhythm disorders and cardiac arrest. Cardiovascular disease is a rare but emerging factor in maternal mortality that can be justified by an upward trend in the mean age of pregnant individuals. Spontaneous coronary artery dissection (SCAD) is defined as a non-traumatic and non-iatrogenic separation of the coronary arterial wall by intramural hemorrhage with or without an intimal tear. The resulting intramural hematoma compresses the coronary arteries, reducing blood flow and causing myocardial ischemia. SCAD continues to be misdiagnosed, underdiagnosed, and managed as an atherosclerotic acute coronary syndrome, which may harm patients with SCAD. The latest research shows that individuals who have or have had coronavirus disease 2019 (COVID-19) may also present coagulation abnormalities, so infection with COVID-19 during pregnancy can increase this hypercoagulable condition, thus increasing the risk of SCAD and SCD. This present study reports two cases over 35 years, one being infected with SARS-COV2 one month before the event and the other being tested positive during admission, both asymptomatic, who were declared healthy on periodic clinical evaluations, with pregnancies over 35 weeks, with normal fetal development, which suddenly caused chest pain, dyspnea, and loss of consciousness, required emergency c-sections, and died suddenly after they were performed. In both cases, the cause of death was SCAD on the anterior-descending artery. In both cases, emergency percutaneous coronary intervention was performed. The second part of the study represents a literature overview of SCAD during COVID-19. In addition to pregnancy hormonal changes, other potential hormone-mediated SCAD triggers are still under discussion. Sudden cardiac death (SCD) represents a non-violent death in the majority of cases with a known or unknown cardiovascular etiology. SCD is defined as death from a cardiovascular disease that occurs within one hour of the onset of symptoms, confirmed by witnesses present, or in less than 24 h without witnesses . It is sCardiovascular disease is an uncommon but rising cause of maternal mortality in both developing and developed countries, which can be explained by an increase in the average age of pregnant women. Despite the difficult screening protocol, observational studies have indicated that mother’s older age at the time of pregnancy is related with an increase in maternal morbidity and death. Still, systematic research for cardiac disease is usually not performed; at an advanced maternal age, investigations focus on prenatal genetic screening and fetal development. As a result, alongside well-established causes of maternal death , undiagnosed cardio-coronary pathologies turn into SCD cases when the autopsy report reveals them.Spontaneous coronary artery dissection (SCAD) is defined as a non-traumatic and non-iatrogenic separation of the coronary arterial wall by intramural hemorrhage with or without an intimal tear. The resulting intramural hematoma compresses the coronary arteries reducing blood flow and producing myocardial ischemia . SCAD inWe report two cases over 35 years old, one being infected with SARS-COV2 one month before the event and the other being tested positive during admission, both asymptomatic, who were declared healthy on periodic clinical evaluations, with pregnancies over 35 weeks and normal fetal development, who suddenly complained of chest pain, dyspnea, and loss of consciousness. They both required emergency coronary stenting and c-sections, and died suddenly after it was performed.A 35-year-old female in apparently good health, pregnant in 34 + 3 weeks with normal fetus development (3 previous pregnancies without problems), without any pathological background or history of alcohol consumption, and a smoker, who checked all her mandatory antenatal visits as normal, was brought to an emergency hospital due to her having sudden chest pain, dizziness, weakness, shortness of breath, and cold sweats, with the diagnosis: “IV pregnancy in 34 + 3 weeks. Viable fetus in cephalic presentation. Intact fetal membranes. Eutocic pelvis. Signs of impending premature labor. Dyspepsia. Hypothyroidism. Tobacco use and addiction”.At the time of admission, laboratory tests revealed SARS-COV2 virus infection through real-time PCR, leucocytosis with neutrophilia, urinary tract infection (nitrite positive), and pregnancy-induced anemia (Hgb 10.6 g/dL). The fetal ultrasonography values were within normal limits. She immediately began tocolytic, hormonal, and antispastic treatment alongside the prevention of hyaline membrane disease. On the second day of hospitalization, the infectious disease consultation stated: “SARS COV2 infection, asymptomatic form. Urinary tract infection” and recommended antibiotic treatment with 3 × 1 g/day of Ceftazidim . 2 saturation of 99%, and angina and minor abdominal pain. She was admitted with the following diagnostics: “Acute myocardial infarction STEMI KILLIP 1, Chronic ischemic heart disease, Hypertension gr. I, Pregnancy 34–35 weeks, SARS COV2 infection” accusing angina. On the third day, the patient complained of angina-type pain accompanied by severe dyspnea. The cardiologist rises the suspicion of an ST elevation anteroseptal myocardial infarction (STEMI)/myocarditis requesting a transfer to a superior county hospital. During the transfer, she presented with a GCS of 15 points, respiratory frequency of 12/min, heart rate of 62 bpm, blood pressure of 113/78, O2 level of 95% without a mask. After 5 h without any symptoms, she suddenly presented with a cardiac arrest with pulseless ventricular tachycardia that responded after 1 min of CPR with brief cardiac activity  and after another minute with a second cardiac arrest. At this point, she is intubated and ventilated; she is administered Adrenaline and after 5 min of CPR, she presents ventricular fibrillation, for which she receives 2 shocks of 200 J each and begins to receive Adrenaline, and 300 mg of Cordarone every 4 min. After 30 min of CPR without a positive outcome, an emergency c-section is decided to save the fetus. She is rushed under CPR maneuvers to the gynecology operating room, where a c-section is performed still under CPR maneuvers without response. Death is declared after 6 h from the initial stenting. The admission EKG stated normal sinus rhythm, a heart rate of 60 bpm, an intermediate QRS axis, and an elevated ST segment in the anterior territory associated with under-leveling of the inferior one. Laboratory analysis found hs-cTnI at 12,000 ng/mL. Echocardiography found undilated left and right ventricles, left ventricular systolic function slightly depressed, EFLV 45%, akinesia of the apex of the septum and left ventricle, second-degree mitral regurgitation, and no free pericardial space. The anterior-descending branch of the left coronary artery was found to have an acute dissection with a 25 mm subocclusive stenosis in II and III segments and TIMI II distal flow. The circumflex coronary artery and the right coronary are both free of angiographic abnormalities. Under the shield of double antiaggregants and efficient anticoagulant use, direct stenting is performed using a DES 3 × 33 mm stent with the re-establishment of the coronary lumen covering the initial part of the dissection, with a good outcome, TIMI III flow being achieved distally. During the interventional coronarography, the patient was hemodynamically stable without inotropic medication, but at the end of the procedure, she complained of abdominal pain, for which an emergency gynecological consult was requested. At this moment, she was without angina, without left ventricular insufficiency, hemodynamically and electrically stable, with a blood pressure of 108/80 mmHg, a heart rate of 60 bpm, and an OAccording to Romanian Law, a medico-legal autopsy was requested by the inquirer . The autopsy revealed a Caucasian female of 167 cm in height and 56 kg in weight. There was no evidence of congenital abnormalities, disease conditions, or trauma, signs of treatment or surgery . All organs were placed in normal anatomical positions; the frontal part of the fundus of the uterus  presenteThe thoracic cavity presented 200 mL of citrine serum liquid in both pleural cavities, with sternal fractures due to mechanical resuscitation. After sectioning the lungs, there were macroscopic signs of acute pulmonary edema . The heart weighed 320 g, measured 13 × 13 × 5.5 cm in dimension, and had a normal valvular aspect. The epicardial adipose tissue was 0.5–0.7 cm in thickness (for the right and left coronaries). Moreover, at the level of the anterior-descending artery, we identified an acute wall dissection with stenosis in segments II and III, as shown in The circumflex artery had a slight hypoplasia (0.3–0.4 cm compared with 0.6 cm of the other coronaries) with no visible atherosclerosis; the anterior-descending artery had a stent of 3 cm with an unobstructed lumen; after removing it, a discontinuity of the wall was observed with hemorrhagic infiltrate surrounding it. Microscopically, as seen in It was concluded that the death was non-violent  due to acute pulmonary edema as a consequence of an acute myocardial infarction from an acute coronary dissection in a SARS-COV2-infected person.A 38-year-old female in apparently good health condition, pregnant at 38/39 weeks, with normal fetus development (one previous pregnancy without problems), with no pathological background, no history of smoking or alcohol consumption, and who checked all her mandatory antenatal visits as normal, suddenly complained of intense chest pain. Five months before the angina attack, she and her child had COVID-19 in asymptomatic forms. She was brought to a county hospital with severe retrosternal chest pain of, and because of the maternal-fetal endangerment, after the cardiological and gynecological consultation, she was transferred to a different county hospital with the diagnosis: “II pregnancy, 38/39 weeks. Viable fetus in the right occipital-iliac cranial presentation. Intact fetal membranes. Eutocic pelvis. The onset of labor. Fibrocicatricial uterus after C-section. Acute anterior myocardial infarction. Chronic ischemic heart disease. Cimented prosthesis of the right hip joint”. Immediately after admission, she underwent an emergency c-section, with a newborn male baby weighing 3930 g with an Apgar score of 7 at 1 min and 9 at 5 min. After the c-section, she was rushed to the interventional cardiology department, where the coronarography performed found acute coronary dissections of the anterior-descending artery with occlusive stenosis and acute dissection of the circumflex branch, for which two stents were mounted and transferred to the intensive care unit of the gynecology department. After this procedure, she was intubated, sedated, and hemodynamically stable, with a well-contracted uterus, under continuous perfusion with Oxistin 10 IU, and diuresis was present (400 mL). 2 level with O2 addition through a mask. The patient’s condition deteriorated abruptly one hour after extubating, with tachypnea, bilateral rhonchi, and pearl-pink sputum as symptoms of severe pulmonary edema. Promptly, she underwent cardiac reassessment and initiated therapeutic intervention comprising Furosemide, Morphine, and non-invasive ventilation (she had an 85% O2 level). Nitroglycerin was not administered due to her hypotension (80/40 mmHg). She did not react to non-invasive ventilation with fast desaturation, so she was intubated and mechanically ventilated, given therapy with Dopamine, and then Dobutamine and Noradrenaline was started. The cardiac ultrasound exam highlights severely depressed left ventricle function with 20–25% ejection fraction, global hypokinesia, akinesia at the apex of the left ventricle, and 3rd-degree mitral valve insufficiency. Without a positive response from the treatment, she presented a clinical pattern of refractory cardiogenic shock, remaining hemodynamically unstable, blood pressure of 65/45 mmHg, a heart rate of 160 bpm, and O2 levels at 65% intubated/mechanically ventilated with 100% O2 added. She underwent another cardiac ultrasound examination, which did not show any improvement from the previous one. A total of 10 h after the stenting, she presented with pulseless electrical activity, and after one hour of CPR with a total of 10 mg of Adrenaline being administered, she was declared dead.She was extubated the next morning, conscious and cooperative after extubation, with a blood pressure of 88/50 mmHg, a heart rate of 115 bpm, and a 100% OA medico-legal autopsy was requested by the inquirer  according to the same law.The autopsy revealed a Caucasian female of 170 cm tall and weighing 72 kg. There was no indication of congenital anomalies, but there was one 20 cm vertical scar with the sign of 19 stitches on the lateral aspect of the left hip, as well as signs of treatment and surgery . All organs were placed in their proper anatomical position; the frontal-upper part of the fundus of the uterus presented a horizontal incision of 13 cm that was constantly sutured , and theThe thoracic cavity had sternal and rib fractures due to mechanical resuscitation. The lungs were heavy, and after sectioning, there were macroscopic signs of acute pulmonary edema and acute stasis. A total of 50 mL of yellowish liquid was found in the pericardial sac. The heart weighed 450 g and measured 12 × 12 × 5.5 cm in dimension, after sectioning, with normal valvular aspect and globally dilated ventricles. The epicardial adipose tissue was 0.6–0.8 cm in thickness (for the right and left coronaries). The dimensions of the ventricular walls were 0.8 × 0.8 × 0.3 cm (left ventricle × septum × right ventricle). On myocardial slides, we observed pale, ischemic tissue, myocardosclerosis as thin white-pearly plots, and in the anterolateral wall of the left ventricle, a diffuse hemorrhagic area of 8 × 1 cm that was visible from the base to the apex .The heart was placed in 10% formaldehyde for one week before being opened to analyze the coronaries. The circumflex artery had hypoplasia from the emergence (0.3 cm compared with 0.6 cm of the other coronaries) and at 3.2 cm suddenly dilated at 0.7 cm with the presence of a thrombus of 2 cm in length with no visible atherosclerosis and a visible thinning of the wall. The anterior-descending artery  had, rigIt was concluded that the death was non-violent  due to acute pulmonary edema as a consequence of an acute myocardial infarction from an extensive acute coronary dissection.SCAD represents one of the rare causes of myocardial infarction and SCD. Like SCD, the etiology, prognosis, and possible treatment of this pathology are not well established, making it a rare but important cause of acute myocardial infarction in young, active patients, especially postpartum women. The true prevalence of SCAD is uncertain because the pathology is often misdiagnosed. Regarding the etiology of SCAD, in the past, DeMaio et al.  underlinFuture research should better define this condition, excluding iatrogenic, traumatic, and atherosclerotic dissection, and describe it as a coronary obstruction caused by the formation of an IMH or an intimal disruption. Recent studies have suggested that SCAD may be the cause of acute coronary syndrome in up to 35% of cases of MI in women aged <50 years and is the most common cause of pregnancy-associated MI ,15. ThisRegarding the coronary distribution of SCAD, any artery can be affected, but the left anterior descending artery is the most commonly affected (32–46% of cases) ,20,21,22In the development of SCAD, two theories are still in debate, both being borrowed from the aneurysm etiopathogenesis: the first, which states that a disruption in the vessel wall  will allow the blood from the true lumen to enter and generate a false lumen , and theSeveral studies ,32,35,36p = 0.013), had left main and multivessel dissections, had lower left ventricle function, and were less likely to have a concurrent FMD.Most pregnancy-related SCAD events occur in the third trimester or early postpartum period, with the left anterior-descending artery being the most commonly affected. It is believed that estrogen and progesterone receptors found in the coronary arteries may weaken the vessel wall, which culminates in coronary wall rupture, IMH, and the onset of clinical symptoms . AdvanceIn addition to pregnancy hormonal changes, other potential hormone-mediated SCAD triggers are still under discussion  ,47,48. BThe traditional angiographic description of SCAD noted the presence of multiple radiolucent lumens and extraluminal contrast staining  . In his Aquaro et al. performed post-mortem cardiac magnetic resonance imaging (PM-CMR) using formalin-fixed explanted hearts from 115 cases of sudden death in their study. When using a guideline-driven histological sample, the PMCMR interpretation matched the final forensic diagnosis in 93 cases (81%) . With the addition of a PMCMR-driven strategy, the matching went up to 102 cases (89%) . The authors found that PMCMR exhibited a high degree of accuracy in identifying the cardiac etiology of sudden death and might be used as a legitimate adjunct for forensic diagnosis . In addiThe American College of Cardiology/American Heart Association and the European Society of Cardiology guidelines for the management of ACS agreed on an early invasive strategy with immediate revascularization of the diagnosed lesions over conservative therapy alone ,60. ThisCoronary guidewires may enter the false lumen and occlude the true lumen. Balloon dilation and stent placement can also increase the risk of extending the intimal dissection in addition to changing the IMH position upstream or, even worse, downstream, producing a distal vessel obstruction that can lead very quickly to SCD.An accurate diagnosis of SCAD will ensure that an invasive strategy of percutaneous coronary intervention (PCI) is reserved only for a properly selected group, given that PCI for SCAD is associated with lower technical success and higher complications than PCI for atherosclerotic disease ,62,63.Despite the latest advances in this topic, SCAD continues to be misdiagnosed, underdiagnosed, and managed as an atherosclerotic acute coronary syndrome, which may harm patients with SCAD. Thus, by the traditional angiographic description of SCAD , >70% of SCAD cases will, unfortunately, be missed. We consider that getting accustomed to the diffuse, narrowed-lumen appearance of IMH and the use of intracoronary imaging may improve the future diagnosis of SCAD.


RNA modifications known as epitranscriptomics have emerged as a novel layer of transcriptomic regulation. Like the well-studied epigenetic modifications characterized in DNA and on histone-tails, they have been shown to regulate activity-dependent gene expression and play a vital role in shaping synaptic connections in response to external stimuli. Among the hundreds of known RNA modifications, N6-methyladenosine (m6A) is the most abundant mRNA modification in eukaryotes. Through recognition of its binding proteins, m6A can regulate various aspects of mRNA metabolism and is essential for maintaining higher brain functions. Indeed, m6A is highly enriched in synapses and is involved in neuronal plasticity, learning and memory, and adult neurogenesis. m6A can also respond to environmental stimuli, suggesting an important role in linking molecular and behavioral stress. This review summarizes key findings from fields related to major depressive disorder (MDD) including stress and learning and memory, which suggest that activity-dependent m6A changes may, directly and indirectly, contribute to synaptic connectivity changes underlying MDD. Furthermore, we will highlight the roles of m6A and FTO, a m6A eraser, in the context of depressive-like behaviors. Although we have only begun to explore m6A in the context of MDD and psychiatry, elucidating a link between m6A and MDD presents a novel molecular mechanism underlying MDD pathogenesis. Epitranscriptomics, also known as RNA modifications, refers to the study of post-transcriptional modifications of RNA molecules. Although chemical modifications of RNA have been described for a half-century, only recently with the advancement of technology have we started to elucidate their functions . To dateMajor depressive disorder (MDD) is a complex, multifactorial illness, constituting one of the most important societal burdens worldwide . While tIndeed, numerous studies, in both mice and humans, have shown that stressful exposures result in long-lasting epigenetic changes via DNA methylation and histone tail modifications . LikewisOthers have done an excellent job of reviewing the roles of m6A in brain plasticity, learning and memory ,19, and Methylation at the N6 position of adenosine is referred to as m6A. It is the most abundant RNA modification, with approximately 25% of mammalian messenger RNAs (mRNA) bearing the mark . m6A is Interestingly, m6A is highly enriched in the brain, with more than 30% of the transcripts in the brain harboring this modification . Indeed,Although cell type-specific m6A profiles in the brain have not yet been generated at large, there is evidence to support that the proteins related to m6A are expressed across all cell types, albeit more highly expressed in the neurons compared to the glial cells . HoweverThe delivery of select transcripts to distal compartments, such as axons and dendrites, is critical in neurons where thousands of transcripts are locally regulated, influencing the production of proteins that affect synaptic organization and transmission. Recent research has shown that m6A modifies mRNAs destined for subcellular transport, specifically those targeted to the synapse. m6A profiles from the synaptosomes isolated from mouse forebrains found that m6A transcripts are enriched for pathways associated with synaptic function . SpecifiAnother mechanism by which local translation is regulated is through m6A eraser function within the axon, the effects of which have been associated with axon guidance, growth, and regeneration ,54. The Taken together, the current literature suggests that m6A participates in the localization of target transcripts within neurons. Moreover, those targeted transcripts include the precursors for synaptic structural proteins which are important for synaptic communication. This implies that m6A may be one of the mediators in synaptic communication.Synaptic plasticity is an essential part of the mechanism of neuronal adaptation. There is growing evidence to suggest that mice with mutations of m6A machinery genes affect the learning and memory processes. For example, the deletion of the m6A writer METTL3 in the hippocampus shows normal synaptic transmission and short-term memory but deficits in long-term memory consolidation . At the Likewise, the downregulation of the m6A eraser FTO and, thus, the increase of global m6A methylation level has been linked to learning and memory. At the basal levels, memory formation causes a short-term reduction in the abundance of FTO, preferentially at the synapse . MoreoveYthdf1-knockout mice rescued the behavioural and synaptic defects [Readers, such as YTHDF1, have been shown to mediate the effect of m6A in learning and memory, most likely through the regulation of translation efficiency. For example, deleting YTHDF1 resulted in learning and memory defects, impaired hippocampal synaptic transmission, and long-term potentiation by promoting the translation of neuronal transcripts . Convers defects . This ch defects . AltogetSynaptic communication, within and across brain regions, is critical for mood regulation and cognitive function and alterations to synaptic function are a key feature of psychiatric disorders, including MDD. Emerging evidence implicates m6A in the molecular mechanisms closely associated with synaptic connectivity and psychiatric disorders. Indeed, recent studies have shown altered m6A profiles in neurodegenerative and psychiatric disorders, including Alzheimer’s disease ,60,61, PAs described above, m6A regulation has an important influence on synaptic plasticity, which has a direct impact on learning and memory, a system highly impacted by depression ,66. OtheMETTL3-mediated m6A regulates neurogenesis and neuronal development by modulating the expression of histone methyltransferase Ezh2 [While adult hippocampal neurogenesis (AHN) remains a controversial topic in humans, some studies have nonetheless shown an effect of AHN on psychiatric disorders with evidence that suggests m6A plays a role. The m6A eraser FTO is highly expressed in adult neural stem cells (aNSCs) and its loss reduces proliferation and neuronal differentiation in mice ,67,68. Tase Ezh2 . METTL3 Dysregulation of the immune and inflammatory response, both peripherally and centrally, is commonly found across psychiatric disorders. Several studies have identified links between m6A and the inflammatory responses of microglia and macrophage, particularly in the polarization toward different phenotypes and inflammation: for example, genes related to microglia phenotypes. Pro-inflammatory-like M1-like, anti-inflammatory M2-like, and unstimulated M0-like are represented by state-specific methylation patterns, suggesting a role for m6A in the pro- and anti-inflammatory responses of the brain . In macrAs described above, a number of the systems that are dysregulated in MDD have been found to be mediated through m6A, but only a few studies have directly investigated the effects of m6A on depression and depressive-like behaviors. To date, many of these studies focused on an m6A eraser, FTO, due to its role in obesity and the strong link of metabolic disorders with depression ,77. IndeThe stress response is tightly regulated by the HPA axis, and it is responsible for translating stress-induced stimuli into hormonal signaling. Repeated stress can lead to increased HPA axis activity, increased levels of glucocorticoids, and disrupted negative feedback regulation, which are the hallmarks of MDD. Several studies have investigated the roles of m6A in response to stress. Acute stress was found to alter gene-expression levels of the erasers FTO and ALKBH5, which in turn regulated global m6A levels in the mice’s brains in a region-specific manner, with the global m6A levels increased in the amygdala and decreased in the prefrontal cortex in the mice . In addiTaking advantage of publicly available datasets, a recent study explored the gene expression level of m6A regulators in depression and depressive-like behaviors . AlthougFTO overexpression in mice resulted in hypomethylated genes enriched in pathways associated with synaptic organization and the regulation of neurotransmitters [FTO-knockout or Mettl3-knockout in the dorsal and ventral hippocampus did not lead to significant changes in anxiety-like behaviors but increases in fear memory [To specifically investigate the impact of m6A on depressive- and anxiety-like behaviors, several studies tested the effects of manipulating m6A regulators in order to observe subsequent behavioral outcomes . A studysmitters . These psmitters . Howeverr memory . Moreover memory . OverallFTO knockout in the VTA increased stress vulnerability and abolished antidepressant activity, while FTO overexpression increased the activity of TCAs [Given the roles of m6A in depressive-like behaviors, it thus makes sense that m6A would play a role in antidepressant response. Indeed, a recent study investigated the potential role of m6A in response to antidepressant treatment. Peng-Fei et al. showed that FTO mediates tricyclic antidepressant (TCA) response in the ventral tegmental area (VTA) of mice.  of TCAs . MoreoveGenome-wide association studies have linked variants in m6A-related genes to the risk of developing MDD and other psychiatric disorders ,83. Givem6A participates in a number of diverse functions in the adult brain that underlie neuronal activity and behavior . It is hAlthough recent literature points to the dysregulation of m6A in MDD and psychiatric disorders, there is a clear need for continued work in this field as we are only beginning to scratch the tip of the iceberg. For example, recent studies in MDD suggest that males and females respond differently to stress at multiple molecular levels. Therefore, studying the sex-specific regulation of m6A in the context of stress could identify the underlying mechanism engaged in the response to stressful events. Future studies using human postmortem brains could help us better understand the roles of m6A in MDD. Moreover, given the diverse cell types in the brain and their distinct roles in MDD, single-cell studies will be necessary to address the importance of m6A at a higher resolution. Future studies should also carefully investigate how m6A contributes to the response to antidepressant treatment, leveraging peripheral samples. Ultimately, these studies will help to identify novel pathways, mechanisms, and targets associated with MDD and move toward better interventions and treatments.


Background soils are frequently utilized as a surrogate to assess pollution levels and environmental risks of heavy metals in Chinese lakes. However, there remains a lack of understanding regarding the reliability and uncertainty of such assessments. Here, we determined heavy metals  in sediment cores from five rural lakes in North China to evaluate the reliability and uncertainty of the assessments using background soils by comparing them with assessments based on background sediments. Comparative studies reveal large uncertainties in the assessments using background soils. Among these metals, uncertainties for Hg and Cd are relatively large, whereas those for the other metals are minor. This discrepancy is due to the considerably higher natural variability of Hg and Cd in soils and sediments in comparison to the other metals. Generally, assessments utilizing background soils underestimate pollution levels and risks of Hg but overestimate those of Cd in these lakes. Despite limited human activities around the lakes, they still received a considerable influx of heavy metals via regional atmospheric transport. Assessments of the nine metals indicate moderate to considerable ecological risks in these lakes. The risks are contributed primarily (78–89%) by Hg and Cd. This study underscores the substantial uncertainties in assessing heavy metal pollution and risks using regional background soils and emphasizes the importance of controlling atmospheric emissions of Hg and Cd to mitigate pollution in rural and remote water bodies in China. Lakes constitute a significant portion of surface freshwater in China and play a crucial role in water supply, flood control, landscape entertainment, and the preservation of regional biodiversity ,2,3. HowSediment is the ultimate sink of heavy metals in lakes, and thus it is widely employed as a geo-marker for assessing heavy metal pollution and environmental risks and for identifying potential pollution sources ,10,11,12However, owing to the lack of geochemical baselines for heavy metals in lake sediments, most previous studies conducted heavy metal pollution and risk assessments by referring to regional background soils as a surrogate ,17,19,21Lakes situated in remote areas are frequently subjected to minimal human activity. They usually contain naturally deposited sediments with limited human disturbance. As a result, these lakes provide an ideal opportunity to examine the pre-pollution conditions of sediments and assess the reliability/uncertainty of heavy metal assessments using background soils by comparing them with assessments based on background sediments. Therefore, five representative lakes situated in relatively remote areas across three provinces in North China  were selIn this study, five typical lakes, Gonghai (GH), Mayinghai (MY), Dali (DL), Zhagesitai (ZS), and Kulun (KL), from three provinces in North China were chosen for investigation . GeneralIn Shanxi Province, two sediment cores were retrieved from lakes GH and MY in 2014 using a gravity core with a 90-millimeter-diameter polycarbonate tube. The other three were sampled from lakes DL, ZS, and KL in 2016. The sediment cores were specifically taken from the lake center areas GH, MY, ZS, and KL with water depths of about ~10 m, ~9 m, 1.8 m, and 4.0 m, respectively, and from a deep water area (approximately 10 m) in lake DL . Some ot2. Quality control was implemented by digesting and analyzing analytical blanks and standard reference materials . The average recovery rates for Hg in the reference material GBW07305 were determined to be 93 ± 8%. For the other sediment samples, mercury concentrations were analyzed using a direct mercury analyzer  following the USEPA method at the State Key Laboratory of Lake Sciences and Environment [−1.Mercury concentrations in the sediment cores from GH were pretreated and measured at the Environment Research Center in University College London, following the method described by Yang et al. (2016) . Initialironment . StandarTo measure concentrations of various heavy metals such as Ti, Zn, Co, Cr, Ni, Cu, As, Cd, and Pb, sediment samples were first digested in Teflon tubes using a combination of hydrochloric acid, perchloric acid, hydrofluoric acid, and nitric acid at temperatures ranging from 145 to 165 °C ,29,30. T137Cs, 210Pb, and 226Ra in the sediment cores were detected with a low-background germanium detector. Specifically, the activities of 210Pb were measured at 46.5 keV, while those of 137Cs were at 662 keV. The activities of 226Ra were detected at 295 keV and 352 keV, and γ-rays were emitted by its daughter isotope, 214Pb [210Pbex supply (CRS) model in combination with 137Cs. The dating details and results can be found in our previous studies by Wan et al. (2020 and 2022) [Furthermore, in order to establish the background (pre-pollution) and present (post-2010) sediments, the activities of e, 214Pb ,31. Morend 2022) ,26. Finai and Xr are concentrations of metal i and the reference element (Ti) in sediment samples and in the background materials, respectively. In this study, background sediments in the five lakes and natural soils in the three provinces  [The enrichment factor (EF) is a widely employed index in assessing the enrichment and pollution levels of heavy metals in soil and sediment, as well as determining their natural and anthropogenic sources ,33,34. Td Hebei)  were conThe EF values allow for the classification of heavy metal enrichments into five categories: (1) EF ≤ 2, low enrichment; (2) 2 < EF ≤ 5, moderate enrichment; (3) 5 < EF ≤ 20, high enrichment; (4) 20 < EF ≤ 40, very high enrichment; and (5) EF > 40, extremely high enrichment ,34.The Nemerow pollution index (NPI) is a weighted multi-factor index used to assess the comprehensive pollution status of all heavy metals present in sediments ,36,37. Tave represents the average value of PI for all the studied heavy metals in a given sediment sample, and PImax is the maximum value of PI among the heavy metals. Ci and Bi denote concentrations of metal i in the sediment sample and in the background material, respectively. It is worth noting that both the NPI and PI can be categorized as follows [In the formulas, PI represents the pollution index (PI) of metal i. PI follows ,36,37: Ni represents the potential ecological risk of metal i in the sediment. Ti  denotes the toxic-response factor of metal i [The calculation for determining the potential ecological risk of metal i in sediment is expressed as follows ,39,40,41 metal i . Csi deni < 40, RI < 150: low ecological risk; 40 ≤ Ei < 80, 150 ≤ RI < 300: moderate ecological risk; 80 ≤ Ei < 160, 300 ≤ RI < 600: considerable ecological risk; 160 ≤ Ei < 320, 600 ≤ RI < 1200: high ecological risk; and Ei ≥ 320, RI ≥ 1200: very high ecological risk.Ecological risk levels associated with heavy metals can be categorized into the following classes ,39,40: EStatistical values of heavy-metal concentrations in present and background sediments in the lakes, as well as in regional background soils, are shown in −1) in GH is only half of that in MY (39.4 ± 1.8 μg kg−1). The large discrepancy in background Hg between the two lakes can also be found in another study by Liang et al. (2022) [−1) in MY background sediments is 1.6 times higher than the soil (24.0 ± 31.1 mg kg−1), whereas that in GH (19.1 ± 2.2 μg kg−1) is only 80% of the soil.In Shanxi Province, the two lakes of GH and MY have roughly similar metal concentrations, except Hg, in their background sediments , corresp. (2022) . Metal cIn the present (2010–2014) sediments, concentrations of As, Cd, Pb, and Hg in MY and of Cd, Pb, and Hg in GH are higher than their background values, while other metals are similar or slightly low . Among t−1) (−1 in DL and 22.3 ± 1.4 μg kg−1 in ZS) in the two lakes’ background sediments are obviously lower than those in the soil (34.0 ± 46.1 μg kg−1).In Inner Mongolia, the two lakes DL and ZS are ~40 km away from each other and both situated in the Hunshandake Sandy Land with similar geological environments . Hence, −1) . In contIn the present (2010–2016) sediments, concentrations of Zn, As, Cd, Pb, and Hg in DL and of Zn, Ni, Cu, Cd, Pb, and Hg in ZS are higher than their background sediments, while other metals are roughly similar. Among the metals, the Hg content in the two lakes’ present sediments is obviously (2.2–3.1-fold) higher than that in their background sediments. The second is Cd, about one-fold higher. Pb and other metals  in the two lakes’ present sediments are slightly (20–30%) higher than those in their background sediments.In Hebei, heavy metal contents in the background sediments of KL are close to those in DL and ZS , as theyi) results for the nine metals in the five lakes by referring to background soils and background sediments. In To evaluate the reliability/uncertainty of pollution and risk assessments of heavy metals in lake sediments using background soils as a surrogate, in the following we compare pollution (EF and PI) and risk (Ei by comparing the two backgrounds (i) and Cd (EF). The assessment results of Hg by referring to background soils are one level higher than those by referring to background sediments.In the two lakes in Shanxi Province, most metals show similar values of EF, PI, and Ekgrounds . RelativIn the two lakes in Inner Mongolia, three metals  show relatively large discrepancies between the two backgrounds . For Cd,In lake KL in Hebei Province, a large discrepancy between the two backgrounds is found in Cd and Hg . For Cd,From From the above comparisons of pollution and risk assessment results of heavy metals in the five lakes from three provinces in North China, it can be found that there are obvious discrepancies in the assessments between the two backgrounds, implying large uncertainties in environmental assessments of heavy metals by referring to regional background soils instead of background sediments in Chinese lakes. Generally, uncertainties for Hg, Cd, and occasionally As are relatively large, whereas those for the other metals are minor. The assessments made by referring to background soils usually underestimate pollution levels and risks of Hg but overestimate those of Cd in these lakes.A direct reason for the uncertainties is the ubiquitous differences in metal concentrations between the regional background soils and the background sediments in the lakes . The wid2) has the largest area among the three studied provinces. There are twelve main soil types and seven parent rock types in the province [2) has the second-largest area among the three provinces. It contains three main soil types: brown soil, tidal soil, and brown soil [2.Among the three provinces, the largest difference in metal contents between regional background soils and sediments is found in the two lakes in Inner Mongolia . This coprovince . The secown soil . The smaAmong the nine metals, Cd and Hg often have the largest uncertainties in pollution and risk assessments when using regional background soils instead of background sediments in these lakes, while other metals show relatively minor uncertainties. For Cd and Hg, their pollution and risk assessment values calculated using background soils are usually one- to several-fold different from those using background sediments in the five lakes. Usually, the assessment values for Cd using background soils are higher than those using sediments, whereas those for Hg are lower. For example, the EF, PI, and RI values of Cd in the two lakes in Inner Mongolia assessed using background soils are nearly three times higher than those using sediments . The larFrom the results (NPI-sediment and RI-sediment) in Among the nine metals, Hg is often the most polluted in these lakes. It usually shows moderately to highly polluted levels with considerable to high ecological risks. In these lakes, Hg contributes 42–71% of the potential ecological risks of all metals . The secAlthough Pb is a polluted metal in the global environment that has attracted special attention in recent decades ,46,47, iThe above environmental assessments suggest that the lakes received a large amount of heavy metals via regional atmospheric transport and deposition, although they are situated far from urban/industrial areas and were affected by limited human activities around them. This fits well with the previous conclusion that these lakes mainly received pollutants via regional atmospheric transport and deposition ,27,29,42In a comparative study of heavy metal assessments in five lakes’ sediments in North China, we found that there are large uncertainties in assessing heavy metal pollution and ecological risks using regional background soils instead of background sediments. Generally, the largest uncertainties are usually found in Hg and Cd, while uncertainties in other metals  are minor. This is caused by large differences in Hg and Cd contents between regional background soils and these lakes’ background sediments. Therefore, in the future, it will be necessary to investigate/obtain real background values of heavy metals in sediments before assessing metal pollution and ecological risks in Chinese lakes. Only in this way can we obtain reliable assessment results and understand the real pollution and ecological risks of heavy metals in lakes, which are helpful for establishing appropriate environmental management countermeasures.In these lakes, the assessment results suggest moderate to high polluted levels of the nine heavy metals in most lakes’ present sediments and moderate to considerable ecological risks. Among the nine metals, Hg and Cd are the major (78–89%) contributors to the ecological risks in the lakes, while the other seven metals all have low ecological risks. The different risks between metals are mainly related to atmospheric heavy metal emissions from anthropogenic sources in the region, implying the importance of controlling atmospheric anthropogenic Hg and Cd emissions in this and similar areas in China.Besides the five studied lakes, Hg and Cd are usually also heavily polluted metals in other Chinese lakes. However, in many previous investigations, they only assessed the pollution and risks of part of the heavy metals in lake sediments, not including Hg and/or Cd. Such assessments would significantly underestimate the pollution and environmental risks of heavy metals in lake sediments. In future studies, investigating heavy metals, including Hg and Cd, is crucial to obtaining reliable pollution and risk assessment results for toxic metals in lakes in China.