Oncorhynchus tshawytscha) sampled from the Susitna River Basin where wild Alaskan Chinook stocks are declining regionally and creating incentives for expanded aquaculture. Our non-invasive population analyses indicated higher levels of observed heterozygosity than expected heterozygosity at the Class I and II loci and minimal genetic differentiation between spatially distinct subpopulations. Class I sequences also showed evidence of balancing selection, despite high rates of non-synonymous substitutions observed, specifically at the peptide binding regions of both MHC genes. Taken together, these results suggest that current management practices for Chinook populations in the Susitna Basin are not likely causing any detrimental biological changes in salmon in relation to immune health. Moreover, the immune health of the hatchery fish, at least in the context of the two populations studied, matches that of the wild fish. Our locus-specific work invites a genome-wide survey such as mapping Single Nucleotide Polymorphisms to better understand the role of the MHC in optimal mate choice and to establish MHC relatedness as a proxy for maintaining genetic variability throughout the entire genomes of fish within populations.DNA sequence variation in the major histocompatibility complex (MHC) is vital to the health of the vertebrate adaptive immune system. Consequently, MHC Class I and II loci have evolved to become the most polymorphic vertebrate genes known. This extraordinary level of variation makes MHC immunity genes both prime indicators of individual fitness as well as highly sensitive biomarkers for assessing the population genetic health of wildlife, and by extension, sustainable management of biodiversity. We examined levels of MHC locus-specific DNA haplotype variation in both wild and hatchery-reared Chinook salmon  are crucial for the fitness of Chinook salmon due to the role they play in disease and pathogen resistance. The objective of this study was to assess the DNA sequence variability among wild and hatchery populations of Alaskan Chinook salmon at the class I α1 and class II β1 exons of the MHC. We hypothesized that the 96 wild samples taken from the Deshka River would display greater levels of observed heterozygosity (Ho) relative to expected heterozygosity (He) in suggesting that individuals with similar phenotypes mate with one another more frequently than would be expected under random mating patterns. Conversely, since no mate selection occurs in the William Jack Hernandez Sport Fish hatchery, we would not expect to see this discrepancy (He = Ho) in the 96 hatchery fish tested in this study. Alternatively, we hypothesized that post-mating selection is driving higher levels of observed heterozygosity as opposed to mate selection. If this is the case, we will observe higher than expected levels of heterozygosity among hatchery salmon. Both populations displayed higher levels of observed heterozygosity than expected heterozygosity at the Class I and II loci but genetic differentiation between the spatially distinct communities was minimal. Class I sequences showed evidence of balancing selection, despite high rates of non-synonymous substitutions observed, specifically at the peptide binding regions of both MHC genes. Oncorhynchus tshawytscha); one occurring in the Deshka River, Alaska, and the other derived from the William Jack Hernandez Sport Fish Hatchery in Anchorage. The prevalence of Chinook salmon declined over the past 50 years [In this study, we examine the genetic variation at the major histocompatibility complex (MHC) in two populations of Chinook salmon , and heart skeletal and muscle inflammation (HSMI) is believed to be largely driven by the aquaculture industry, as it plays the biggest role in the transport of this virus due to its congregation in larger groupings and at higher densities than wild salmon populations . AtlantiGenomic DNA was extracted from axillary fin clips sampled from 96 wild individuals and 96 hatchery-derived Chinook salmon from the Deshka River Weir and the William Jack Hernandez Hatchery in Southcentral Alaska . Isolatiw/v) gel to visualize target-locus amplification quality and controls for contamination. Examples of successful PCR reactions are indicated in PCR primer sequences for each MHC locus (class I α1 and class II β1) are summarized in Population genetic tests for Hardy–Weinberg equilibrium, linkage disequilibrium, Analysis of Molecular Variance (AMOVA), average heterozygosity, and F-statistics were carried out using standard population analysis software applications bundled in GenAlEx v.6.5  and ARLESTRUCTURE v 2.3.4  was usedWe used the Datamonkey HyPhy ,25,26 prA total of 270 aligned original protein coding sequences for 126 PCR products of the MHC class I-α1 locus and 144 PCR products of the MHC class II-β1 locus sampled from 192 individuals were deposited into Genbank (see Data Availability Statement below for Accession Nos.) Among all individuals, there were 34 alleles found among the MHC Class I A1 loci  and 34 pThere was considerable differentiation in the overall variance components present in the two loci due to the discrepancies in alleles present among each class, as well as overall sequence variation  and wild  populations for the Class I loci as shown in p < 0.1) among A1 sequences (Aligned sequences were examined for evolutionary signatures present within sequence data using a multimethod approach with programs bundled in the software packages Single-Likelihood Ancestor Counting (SLAC), Fast Unconstrained Bayesian Approximation (FUBAR), and the Mixed Effect Model of Evolution (MEME). These programs were implemented as grounds for identifying pervasive diversifying or purifying selection (SLAC and FUBAR), and episodic diversifying selection (MEME) present within wild and hatchery sequences. Each methodology analyzed 142 sequences at 76 sites (Class I A1) and 145 sequences at 73 sites (Class II B1) for the two MHC genes under investigation. The MEME methodology  revealedequences . The SLAequences . There aequences  present equences .Rates of substitution were much higher at putative peptide binding regions than in non-binding codons . PBR codSalmo salar ; Oncorhynchus gorbuscha ; and Oncorhynchus mykss (Rainbow trout) were also added to display haplotypic clustering and genetic variation among populations.Aligned sequences were analyzed using the STRUCTURE (v. 2.3.4) software to assess inferred ancestry of individuals for both the MHC Class I and II loci. STRUCTURE identifies subpopulations of samples by detecting differences in allele frequencies among samples. Salmo salar; Oncorhynchus mykiss; Oncorhynchus gorbuscha) are indicated by predominantly yellow-coded bars; and British Columbian lineages are indicated by predominantly purple or light blue-coded bars [Distinct clustering can be seen in the STRUCTURE plot assuming six populations  where hoded bars ,4.Using the median-joining network method, phylogenetic maps created in PopART (version 1.7) show the distances of each allele, and their differentiation from their most closely related individual (indicated by hash lines on each connecting arm). PopART allows for visualization of intraspecific relationships and haplotype inference. The central web of alleles is only differentiated from two of the connecting arms by one variation in the amino acid sequence but show considerable differentiation from the Evans and Miller consensus sequences  ,4. TheseOncorhynchus mykiss; Oncorhynchus gorbuscha; Salmo salar; Miller sequences (Onts-A1*1)) [Oncorhynchus phylogeny. Nucleotide diversity among the sequences was 0.06, where 36 of the 56 segregating sites were identifiable as parsimony-informative sites.Using the TCS network method, the Class I haplotype network shows a more substantial amount of variation. The central web (s-A1*1)) ,4 includThe main objective of this study was to test competing hypotheses determining the role of mate selection and inbreeding avoidance as drivers of increased heterozygosity among hatchery and wild Chinook salmon populations in Southcentral Alaska. It also serves as a comparative analysis of genetic variability retention among wild and hatchery populations, and as a measure of genetic and overall health. Additionally, it provides a comparative analysis of genetic variability retention among wild and hatchery populations. The findings of this study underscore the high level of genetic variability present among MHC Class I genes in both sample populations of Chinook salmon while also revealing lower-than-anticipated levels of polymorphism found in the MHC Class II gene that has been observed in previous studies of Chinook salmon . Tests oPoecilia reticulata) by Fraser and Neff [Salmo trutta) determined that individuals retaining rare alleles displayed better fitness regardless of zygosity, arguing that balancing selection was driven by a rare allelic advantage instead of heterozygote/divergent allelic advantage [This could mean that there are unique selective pressures between the two classes of MHC genes at the sequence level. This could also be a product of a comparatively smaller sample size  than utilized in similar studies ,3,4. A sand Neff  stated tdvantage . UltimatThe presence of particular alleles was generally not relegated to either population of Chinook salmon in this study as multiple alleles were present within both hatchery and wild individuals. All four alleles were found among hatchery fish at the Class II B1 locus and three of the four were present in the wild stock. In total, Twenty-seven of the 34 MHC Class I alleles were present within the wild stock and 14 were present among hatchery salmon. Other studies on salmonid species have shown higher rates of allelic diversity . The Landry and Bernatchez  study ofThis pattern was also reflected by the statistical analyses of population structuring among the sample treatments. STRUCTURE (2.3.4) plots partitioned Class II genetic variants into subpopulations based on patterns of similarity. Of the 14 individuals denoted as being derived ancestrally from population 2, 11 were hatchery and 3 were wild. Thus, although there was inferred clustering based on genetic differentiation, it was not isolated to one population. Similar clustering was seen at the Class I locus. Of the 51 individuals clustered into population 2, 40 were wild stock and 11 were hatchery. The discrepancy here is likely a result of differentiation in the final number of individuals used for each calculation (86 wild vs. 48 hatchery) but shows that these alleles persist across geographically divergent lineages. The results of the PopART haplotype networks reiterates the presence of intermixed genetic ancestry among the sampled individuals. While there appears to be distinct haplotype clustering among the samples, the hatchery and wild subpopulations are not genotypically mutually exclusive.Reviewing the results of the AMOVA conducted for the Class I and II loci , it cp < 0.1), but there is evidence of additional sites (13 and 55) that may be under weaker selective pressures throughout the amino acid sequences  but not significant for the hatchery population (p = 0.14). For the Class II loci, the hatchery population had an overall nucleotide diversity of 0.004, with only four segregating amino acid sites. Tajima’s D was −1.17. Wild populations had a nucleotide diversity of 0.002 with three segregating sites. The Tajima test statistic for this population was −1.48. In instances of a negative Tajima’s D, heterozygosity is lower than would be expected (He > Ho). This means that there is an abundance of rare alleles present within a population and may be reflective of population expansion after a genetic bottleneck, or a selective sweep of a beneficial allele resulting in it being fixed within a population  [p) = 0.09 for rejecting the null hypothesis of strict neutrality (dN = dS) among hatchery individuals [p-value of (p) 0.13 for the Nei–Gojobori test of neutrality.Rates of genetic mutations are reflected in the various methodologies  utilized to measure non-synonymous and synonymous substitutions present within the sequences. The SLAC-methodology-calculated dN/dS ratios in equences . The FUBequences  present equences . Finallylection) . The Neiividuals . The wilThe prevalence of mutation was considerably higher in regions of the PBR comparative to that of non-coding regions. Sites of positive selection were predominantly found at loci related to the PBR, suggesting that amino acids of the peptide binding region are undergoing selection that favors diversity for Class I and II loci. In theory, the more polymorphic these sites are, the greater the defense against parasites and pathogens the individual, its offspring, and the overall population should have. Previous studies echo these findings  and go oRhodeus ocellatus), it was determined that female mate preference coincided with the genetic compatibility of the male [There are many vertebrate species that utilize olfactory cues as a method of identifying mates with dissimilar MHC genes, which is identifiable through scent . The devthe male . This gethe male . Thus, ithe male . A studythe male . The offthe male . The hatthe male . As is tSeveral studies have suggested that salmonid females mate non-randomly at MHC loci, preferring mates that offer greater genetic variability at these loci, but randomly on the basis of relatedness through post-copulatory selection ,50. ConsBoth populations exhibited an excess of heterozygotes for the MHC Class I and II loci suggesting selection favoring heterozygous individuals at the MHC. This also lends support to the alternative hypothesis that post-mating selection is driving high levels of heterozygosity among hatchery and wild fish. This suggests that Chinook salmon target mates based on complimentary MHC genes to increase the fitness of their offspring, particularly at the PBR of MHC genes. The information relayed in  suggestsBased on the findings of the statistical analyses discussed above, it can be determined that while high levels of non-synonymous substitutions are occurring at PBR codons in both genes, they remain under balancing selection. Measures of dN/dS revealed a highly polymorphic MHC Class I gene, but based on Tajima’s D statistic, it is not likely that this gene is evolving in a manner that rejects neutrality. However, it would appear that the benefit of heterogeneity at the PBR is pushing the expanded allelic diversity at this locus. Having a high dN/dS value at the PBR codons suggests that this region is undergoing selection that favors amino acid diversity resulting in an advantageous sweep of genetic variants. This suggests that the Chinook salmon studied here should be able to mount a substantial immune response to a variety of endogenous pathogens. The CD8+ T cells produced by the MHC class I genes serve as mediators of adaptive immune systems through immunosurveillance that result in the elimination of tumor cells, virally infected cells, and intracellular pathogens . The ClaUsing a medley of statistical analyses, this study demonstrated high genetic variability is being preserved among MHC Class I and II genes and highlighted the selective pressures that are driving such high levels of variation. Levels of dN/dS showed high rates of non-synonymous substitutions at the PBR codons found in MHC genes but not at overall levels that would reject neutral evolutionary theory. In other words, the changes occurring at these genes are not likely to be causing any detrimental biological changes in the salmon in relation to immune health. The salmon originating from the William Jack Hernandez Hatchery show similar levels polymorphism to those of the wild stock from the Deshka River at both MHC loci. This bodes well for the hatchery practices utilized by the state of Alaska and shows the importance of championing genetic composition throughout hatchery applications but only focuses on one piece of the puzzle. Although the MHC genes studied here have maintained healthy levels of diversity, this study does not encompass the entire genomic health of the fish.As such it invites a genome-wide study such as mapping Single Nucleotide Ploymophisms (SNPs) that could further our understanding of the role of MHC haplotypes in mate choice while also providing new biomarkers for optimizing genetic diversity and adaptive resiliency of fish produced by aquaculture programs targeting Chinook salmon.


Sapindus is an important forest tree genus with utilization in biodiesel, biomedicine, and it harbors great potential for biochemical engineering applications. For advanced breeding of Sapindus, it is necessary to evaluate the genetic diversity and construct a rationally designed core germplasm collection. In this study, the genetic diversity and population structure of Sapindus were conducted with 18 expressed sequence tag-simple sequence repeat (EST-SSR) markers in order to establish a core germplasm collection from 161 Sapindus accessions. The population of Sapindus showed high genetic diversity and significant population structure. Interspecific genetic variation was significantly higher than intraspecific variation in the Sapindus mukorossi, Sapindus delavayi, and combined Sapindus rarak plus Sapindus rarak var. velutinus populations. S. mukorossi had abundant genetic variation and showed a specific pattern of geographical variation, whereas S. delavayi, S. rarak, and S. rarak var. velutinus showed less intraspecific variation. A core germplasm collection was created that contained 40% of genetic variation in the initial population, comprising 53 S. mukorossi and nine S. delavayi lineages, as well as single representatives of S. rarak and S. rarak var. velutinus. These results provide a germplasm basis and theoretical rationale for the efficient management, conservation, and utilization of Sapindus, as well as genetic resources for joint genomics research in the future. The perterpenes . More thterpenes  and showterpenes , elutionterpenes , pharmacterpenes , and phyterpenes . Saponiny washes , seeds oin China  with ann threats . Hence, ollected . HoweverGermplasm resources form the foundation of forest genetic breeding, and the development of forest tree breeding and industry depends largely on the extent and diversity captured by these resources. However, redundancy in germplasm resources may lead to lower conservation and management efficiency. The construction of core germplasm collection is the optimal solution to genetic redundancy. Core germplasm collection is a subset of germplasm accessions that represents the minimum repeatability and maximum genetic diversity of one species . They haDendrobium officinale  markers, have been widely applied in genetic breeding, variety identification, germplasm diversity evaluation and conservation . EST-SSRficinale , Paeoniaa rockii , coconuta rockii , and Stebaudiana  and to cxburghii , crape mxburghii , and olixburghii . Previoupulation , howeverSapindus individuals. The aim was to obtain a representative core germplasm collection which would retain maximum amount of genetic diversity and population structure of the sampled Sapindus population. The core germplasm will enable more scientific and rational conservation, management, and utilization of the genetic resources in Sapindus.In this study, 18 EST-SSR markers were selected based on whole transcriptome sequencing and used to evaluate the genetic diversity and population structure of 161 Sapindus whole transcriptome sequencing project using MISA1 , comprising 175 clones and 64 half-sib families by grafting and seeding respectively, with at least 15 plants for each clone. The average temperature in germplasm nursery is 17.4°C, with a maximum temperature of 36.8°C (July) and a minimum temperature of 4.3°C (January), and an average relative humidity of 83.9% . The DNA samples concentration and quality were determined with a VERTEX 70 UV spectrophotometer .300 mg fresh leaf tissue from each Sapindus tree samples. The forward primer of each marker was labeled at the 5′ end with a fluorescent dye  according to the target fragment size of the marker. PCR (Polymerase Chain Reaction) was performed on a T100 thermal cycler (Biorad) in a 20-μL volume that contained 10.0 μL 2×Taq Plus PCR mix , 0.3 μL 10 μM forward primer, 0.3 μL 10 μM reverse primer, and 1.0 μL genomic DNA. The PCR protocol was 5 min denaturation at 94°C; 20 cycles of 30 s at 94°C, 30 s at 52/72/95/50°C  and principal coordinate analysis (PCoA) were also performed in GenAlEx v6.5 combined with Microsoft Excel. A neighbor-joining (NJ) tree was generated based on pairwise genetic distances between individuals by using PowerMarker V3.25 , number of effective alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), unbiased expected heterozygosity (uHe), ibreeding coefficient (F), and Shannon’s information index (I) were calculated using GenAlEx v6.5 . The poler V3.25 , and ploK value) ranged from 1 to 20 with 10 replicate runs for each analysis. The optimum K-value was determined by the highest ΔK method  was then selected as the optimal core germplasm collection  and lowest for S. mukorossi versus S. delavayi (0.122).There were marked differences in genetic diversity among the four different dus taxa . S. mukoas 0.154 ; highestSapindus was split into three clusters on the first principal coordinate axis (PCoA 1), which corresponded approximately to the S. delavayi group, the S. rarak and S. rarak var. velutinus group, and the S. mukorossi group. On the second principal coordinate axis (PCoA 2), individuals of S. mukorossi were roughly divided into two subgroups: the individuals from southern Guizhou province and the remaining S. mukorossi germplasm. Likewise, S. rarak and S. rarak var. velutinus were also divided into two subgroups along the second principal coordinate axis.The first and second coordinates of the PCoA analysis accounted for 40.54 and 13.44% of the total genetic variation, respectively . The popSapindus population could be genetically divided into two distinct subgroups by the ΔK method . Intriguingly, individuals from southern Guizhou province formed a separate subgroup (K = 3–6), suggesting that they originated from a distinct ancestral population.In contrast to the PCoA results, the STRUCTURE analysis indicated that the K method . Subgrousubgroup . FurtherS. mukorossi, S. delavayi, and S. rarak into their own populations, except for one S. delavayi individual that was grouped together with the S. mukorossi individuals. Furthermore, S. mukorossi individuals could be divided into multiple subgroups which largely correlated with their geographic distribution. Interestingly, some S. mukorossi individuals from Guizhou province appeared to be more closely related to S. rarak.The neighbor-joining dendrogram based on genetic distances among individuals  grouped P ≤ 0.05) and highly significantly (P ≤ 0.01) from the full population in Ne and I, respectively. The core 10–25% subgroups differed highly significantly (P ≤ 0.01) and significantly (P ≤ 0.05) in Na from the core 30 and 35% subgroups. The remaining parameters showed no significant differences between the subgroups and the full collection. Hence, the core 40% subgroup was selected as the optimal core germplasm collection. It contained 64 Sapindus individuals: 53 S. mukorossi, nine S. delavayi, one S. rarak, and one S. rarak var. velutinus ; S. rarak and S. rarak var. velutinus showed less variation   , 3. ThisS. mukorossi, S. delavayi, S. rarak, and S. rarak var. velutinus in nature because of their high phenotypic similarity, and there has been a lack of molecular biological support for the species splits in the Sapindus taxonomy. A better understanding of Sapindus genetic structure is a first step toward addressing these issues. Here, we have identified varying levels of genetic divergence among S. mukorossi, S. delavayi, S. rarak, and S. rarak var. velutinus for the first time using SSR markers. Pairwise FST comparisons among the four Sapindus taxa showed high genetic differentiation   , 17.96% ticulata , 35%  and indel markers that can be obtained by whole genome resequencing or transcriptome sequencing, the number of EST-SSR markers in this study is relatively low, and the results obtained are not sufficient to fully elucidate the genetic structure of Sapindus. Hence, in future studies we hope to perform transcriptome sequencing or resequencing of this core germplasm collection to further explore interspecific and intraspecific genetic structure, evolutionary history, and regions under natural selection in Sapindus. Nevertheless, we believe the results of this study can help facilitate the efficient management, conservation, and utilization of Sapindus germplasm resources in the future.In this study, we selected a 40% (64/161) subgroup of the ividuals . During rameters . Comparerichness , showingrichness . Previourichness . The comSapindus germplasms using 18 EST-SSR markers. Interspecific genetic variation was significantly higher than intraspecific variation in S. mukorossi, S. delavayi, and S. rarak plus S. rarak var. velutinus populations. S. mukorossi had abundant genetic variation and showed a pattern of geographic variation, whereas S. delavayi, S. rarak, and S. rarak var. velutinus showed less intraspecific genetic variation. A core germplasm collection was defined as 40% of the initial population; it comprised 53 S. mukorossi, nine S. delavayi, one S. rarak, and one S. rarak var. velutinus. The results obtained here provide a germplasm basis and theoretical rationale for the efficient management, conservation, and utilization of Sapindus germplasm, as well as genetic resources for joint genomics research in the future.In this study, we have revealed high genetic diversity and significant genetic structure in 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/Sapindus mukorossi. JL, SG, MW, and XW collected the samples. JL, SG, and MW performed molecular labwork and scored the markers. JL, JN, NR, and JY analyzed the data. JL wrote the manuscript with input from JS, and feedback from all the authors. LJ contributed to the special foundation for National Science and Technology Basic Research Program of China and National Natural Science Foundation of China. JL contributed to the China Scholars Council. JS contributed to the Academy of Finland. All authors read and approved final manuscript.JL, SG, LJ, and JS conceived and designed the project. YX completed the transcriptome sequencing of XW was employed by Yuanhua Forestry Biological Technology Co., Ltd. The remaining 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 prognosis for female patients with locoregionally recurrent breast cancer has improved with the concurrent local and systemic treatment under multiple disciplinary teams. Radiotherapy is a valuable local treatment measure for unresectable locoregional recurrent breast cancer; however, reirradiation in previously irradiated areas is still a matter of debate. Antihormonal therapy achieves an overall survival benefit for most of these patients with estrogen receptor-positive (ER+) breast cancer in both adjuvant and metastatic settings. Fulvestrant is an ER antagonist and selective ER downregulator widely used in antihormonal therapy, especially in recurrent postmenopausal ER+ breast cancers. However, fulvestrant closely resembles 17β-estradiol in its molecular structure which may result in false increases in serum 17β-estradiol levels in commercially available immunoassays leading to incorrect medical decisions. Herein, we report a case of a 57-year-old postmenopausal patient with recurrent ER+ breast cancer treated with concurrent fulvestrant and reirradiation. There was a good clinical response, and the combination treatment was well tolerable. During the quarterly follow-up, we monitored a gradual increase of the serum 17β-estradiol level in immunoassays, unexpectedly, because the patient underwent natural menopause 8 years ago. To rule out the suspected fulvestrant cross-reactivity with 17β-estradiol in immunoassay, the patient’s serum 17β-estradiol levels were subsequently tested with the more sensitive and specific liquid chromatography-mass spectrometry (LC-MS) method, which confirmed 17β-estradiol levels at the postmenopausal level. Concomitant fulvestrant with reirradiation seems to be a safe and effective therapy for locoregionally recurrent ER+ breast cancer. However, a falsely increased 17β-estradiol may result from cross-reactivity between 17β-estradiol and its molecular analog compounds, for example, fulvestrant. Therefore, it is important for the clinicians with the knowledge of this interaction to prevent unnecessary erroneous interpretation of results and avoid wrong medical decisions. Breast cancer is one of the most common malignant tumors in women and the leading cause of cancer-related death, approximately 70% to 80% of which are estrogen receptor-positive (ER+) , 2. SincHowever, fulvestrant closely resembles 17β-estradiol in its molecular structure which may result in a false increase in serum 17β-estradiol levels in commercial immunoassays leading to incorrect medical decisions –12. HereDuring the course of the follow-up, we monitored a gradually increased serum 17β-estradiol level in immunoassay. This finding was puzzling because the patient reported menopausal 8 years ago. To further clarify the unexpectedly increased 17β-estradiol level, the patient’s serum 17β-estradiol levels were subsequently obtained and tested with a more sensitive and specific liquid chromatography–mass spectrometry (LC-MS) method , 14. Thein situ hybridization (FISH), and Ki67 positive in 40% tumor cells. We offered a thorough examination to exclude any distant metastases. Laboratory exams were unremarkable before commencement of concurrent fulvestrant with reirradiation treatment, including tumor markers and a reproductive hormone profile. Serum levels of AFP, CEA, CA125, and CA153 tumor markers were 4.00 ng/ml, 1.03 ng/ml, 14.90 U/ml, and 6.60 U/ml, respectively  of the left breast. The pathology revealed IDC, moderately differentiated (G2), measuring 3.5 * 2 * 1.5 cm. There was 1 of 18 lymph nodes that had macrometastatic disease. Immunohistochemical (IHC) examination showed the presence of both ER and PR in 80% of tumor cells with strong staining, while HER-2 showed negative, and Ki67 positive, in 20% tumor cells. The pathologic stage was determined to be pT2N1 (stage II) according to the AJCC staging system. The patient received postoperative adjuvant chemotherapy consisting of 4 cycles of triweekly epirubicin and cyclophosphamide, then 4 cycles of triweekly paclitaxel followed between June 2013 and December 2013. She also received postmastectomy radiotherapy (PMRT), with a total dose of 50 Gy at 2 Gy/fraction × 25 fractions to the chest wall and the supra-/infra-clavicular region (SCN), with intensity-modulated radiation therapy (IMRT) and 6-MV photon. PMRT was completed in January 2014. Antihormonal therapy with highly selective non-steroidal aromatase inhibitor anastrozole was given regularly after confirming her postmenopausal status without interruption. The patient completed her 5 years of antihormonal therapy in January 2019.For this first locoregional recurrent breast cancer, given the patient refused surgical treatment, concurrent fulvestrant (500 mg intramuscular injection every 4 weeks) with reirradiation as a palliative treatment was proposed under our multiple disciplinary team (MDT). The scheduled radiation dose was 60 Gy in 30 fractions with 6-MV X-ray delivered by 3-dimensional conformal radiation therapy (3D-CRT) conducted to the primary tumor volume  offers superior analytical sensitivity and specificity compared to immunochemical methods , 14. TheAfter reviewing the available literature, we found that there were quite a few reports on the falsely increased 17β-estradiol levels and the cross-reactivity between antihormonal agents and standard commercially available 17β-estradiol immunoassays. Fulvestrant sharing the basic structure of 17β-estradiol has also scarcely been reported to cause falsely increased 17β-estradiol levels or cross-reactivity with 17β-estradiol immunoassays. The case presented here may help clinicians to differentiate the falsely increased 17β-estradiol levels to avoid wrong medical decisions.Concomitant fulvestrant with reirradiation seems to be a safe and effective therapy for unresectable locoregional recurrent ER+ breast cancer. Serum 17β-estradiol levels reflecting menopause status may affect treatment decisions for patients with ER+ breast cancer. However, a falsely increased 17β-estradiol level may result from cross-reactivity between 17β-estradiol and its molecular analog compounds. For patients receiving fulvestrant, spurious results may be generated that could impact treatment decisions, so LC-MS is strongly recommended in this setting. It is also important for the clinicians to know this interaction to prevent unnecessary erroneous interpretation of results and wrong medical decisions.The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding author.Written informed consent was obtained from the patient for the publication of any potentially identifiable images or data included in this article.JD conceived the idea of the case report. JD, YC, and YG were involved in the patient’s clinical management. JD performed the literature search and drafted the manuscript. YC designed and supervised the study. All authors contributed to the article and approved the submitted version.This work was supported by the Natural Science Foundation of Jiangxi Province, No. 20171BAB205057.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.


No datasets are available on the basis of confirmed familial breast cancer patients and their respective mutation status for the South African Indian population. This dataset contains information collected in South Africa specifically for the South African Indian population (n = 223). The data involved a combined collection of laboratory confirmed genetic testing results, accompanied by immunohistochemical and anatomical pathology results. Various patient self-identified variables were also in measured. All the immunohistochemistry and anatomical pathology results were confirmed by a trained medical pathologist. Furthermore, mutation status and the detection of either benign polymorphisms or variants with an unknown clinical significance at the time of data collection were also reported. This dataset contains variables in a tabular format which includes the accompanying variables for each patient. All the patient-specific data were deidentified which allows for further exploration of the data with respect to the demography, immunohistochemistry and genetic material. This dataset has the potential to serve as the backbone for translational research and inquiries into this unique South African population. Furthermore, in the final dataset, only patients classified from the South African Indian population were used. The data was stratified into three overarching components namely: demographic insights, clinical evidence and genetic information. The final dataset (which includes the genetic screen and the data from the patient request form) is presented in a raw tabular data format, and consist of demographics data (prefix d), clinical- (prefix c) and genetic variables (prefix g), all of which are aimed to provide a clear assessment of what is needed to make an informed diagnosis for familial breast cancer in this population group. All of the variables within the dataset were categorical, and the validity of each variable was confirmed by either a medical officer, medical specialist or medical scientist. The demographic data is what was identified at time of first consultation . Apart fThe clinical data is a combination of family-specific clinical questions aimed at determining disease risk, in combination with confirmed immunohistochemistry and anatomical pathology results for each patient . For somBRCA1/2 genes BRCA1/2 genetic variants were annotated according to specified international nomenclature using the NCBI chromosomes and transcript reference sequences for each of the genes . The genetic data in the dataset is unique to the South African Indian population. Within the dataset, the type of genetic result, if the genetic result caused a frameshift mutation, deletion, or a premature termination was included. There are genetic variations that were classified as variants with an unknown clinical significance (VUS). These variants did not at the time of publication have a known clinical significance and these variants are still under investigation. In addition to this, if the genetic change was as a result of a shift from either a purine to a pyrimidine (or vice versa), or no change – then it was reported for each nucleotide combination.The genetic information is based on the laboratory-specific test results for each of the patients . All of The categorical parameters in this dataset contains a variety of different variables related to clinical genetic information specific to the South African Indian population.2BRCA1/2 variant, the patient is referred for genetic testing. Genetic testing generates the data related to the genetic features in the dataset. This entails the extraction and purification of DNA from blood representative of a specific patient. Once extracted, analysis is conducted using a series of investigative laboratory procedures, to collectively detect all the various types of potential variants, such as single nucleotide changes or copy number variants. All tests are founded on the exponential multiplication of highly specific areas in each of these genes to identify changes on both DNA and protein level. If any anomaly is detected , it will be scrutinized using various methodologies, including DNA sequencing, to confirm its presence. Once the presence of an anomaly is detected, the information is captured. The genetic screening process is broken up into several stages due to the large size of BRCA1 and BRCA2   .Fig. 2DaThere are instances where all processes occurred simultaneously . Under such circumstances, there will be no supporting data from the pathology results captured on the patient request form. In such an event where the first two tests were conducted prior to the request for genetic testing, the clinical information will accompany the clinical request form, where after genetic testing will commence. Once a pathogenic anomaly is confirmed, the information is stored in a secure database. This database contains all the relevant information about the patients that came from the patient request form as well as all the genetic data that was generated and confirmed through Sanger sequencing. Some of the variables and variable fields undergo feature engineering steps. These steps are introduced to deidentify the samples and stratify the information according to the experiments conducted. Each of the patients in the study underwent a further clinical justification step according to their risk profile which was further stratified into low, moderate, and high risk. At every step of the process, all the information was signed off by a senior medical scientist or medical specialist. The data is also collected and validated at each step of the data collection cycle by two medical scientists .Ethical approval for the study was obtained from the Health Sciences Research Ethics Committee at the University of the Free State (ECUFS 107/2014) and the Department of Health, KwaZulu-Natal (HRKM194/14). All the patients in this study completed an informed consent form. Only patients that allowed for further analysis of their samples were included in the dataset. Within this study and with this dataset there were no (none) patients that objected to the storage, study and follow-up analysis using their information. All of the procedures and standards of this study followed the strict and clear protocol of the project plan, under the guidance of the ethical measurements used in this study.By adhering to all the ethical guidelines, we confirm that informed consent was obtained for all participants in this study. The research in this study was carried out in accordance with the code of ethics from the South African Medical Research Council, the University of the Free State Medical School Ethics committee, and the Department of Health, KwaZulu-Natal which are in accordance with the World Medical Association's Declaration of Helsinki.H.M.V.E. Combrink: Conceptualization, Methodology, Formal analysis, Writing – original draft. N.C. van der Merwe: Resources, Conceptualization, Validation, Data curation, Writing – review & editing. R. Katarya: Conceptualization, Methodology. K. de Wet: Writing – review & editing, Writing – original draft. M.H. Motloung: Writing – review & editing, Writing – original draft.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:The authors declare that they have no known competing financial interests or personal relationships that could appear to influence the work reported in this paper.


We investigated the awareness, perceived usefulness, and use of genetic testing among Hispanic and Latino individuals. Annual follow-up surveys for the Hispanic Community Health Study/Study of Latinos (HCHS/SOL) from 2019 to April 2020 assessed participants’ level of awareness and use of genetic tests to determine disease risks, likelihood of passing disease to children, disease treatment, or drug selection. They also were asked to rate the usefulness of the tests for managing a person’s health on a 1  to 10 (extremely useful) scale. There were 5,769 HCHS/SOL participants who completed at least one survey question. Of the target population, 55.2% was aware of at least one type of genetic test. Awareness varied between HCHS/SOL enrollment sites and was higher among individuals who had higher educational attainment and had higher incomes. Only 3.3% of the target population reported receiving one or more of the tests described. HCHS/SOL individuals rated the usefulness as 8.4, on average, with lower scores observed among U.S.-born individuals compared to individuals born outside the United States, with differences by HCHS/SOL enrollment sites. In conclusion, while awareness of genetic testing among Hispanic and Latino individuals varies by location, education, and income, perceptions about its usefulness are high while experiences with testing are rare. Results identify groups and locations that may benefit from greater outreach about the capabilities of genetic testing and precision medicine. Hispanic and Latino individuals who are unaware of genetic testing may not benefit from genomic medicine improvement. Our survey of the HCHS/SOL participants showed low use of genetic tests despite positive usefulness perceptions, and found awareness of genetic testing varied by location, education, and income. Already, genomic testing is accelerating diagnoses and identifying individuals with genetic predispositions for highly actionable conditions.,,,,,,,,,,Analyses of genomic testing show uneven uptake between racial and ethnic groups, including Hispanic and Latino populations.,,,Attitudes toward genomic testing, including perceived usefulness, may also influence the use of genetic tests. Perceived usefulness is a key component of numerous health behavior theories, including the health belief model, protection motivation theory, and adaptations of self-regulation theory to address genetic service useWe addressed this lack of data by surveying participants of the Hispanic Community Health Study/Study of Latinos (HCHS/SOL).The HCHS/SOL is a population-based longitudinal cohort study established to study risk and protective factors in cardiovascular disease development among Hispanic and Latino individuals in the United States. Details of the HCHS/SOL study design and cohort have been reported previously.Between April 2019 and April 2020, the HCHS/SOL AFU survey included items about genetic testing awareness, use, and perceived benefits  and S2. The survey was administered in either English or Spanish, depending on participant preference. Survey items are presented in the Appendix.We extracted fixed and time varying covariates from the data collected at the most recent HCHS/SOL examination available for each individual. Covariates included age at the time of survey completion , gender, study center , self-reported Hispanic or Latino background , educational attainment , household income level , current health insurance status, number of doctor visits within the prior year at the baseline visit , employment status , nativity (born in the 50 U.S. states or not), and marital status . Marital status was available only from the baseline exam and second AFU year (7–9 years before the genetic testing survey items were administered). Although a question about self-identified race was administered to HCHS/SOL participants, it was not used in the analyses because the majority of HCHS/SOL participants refused to answer the question or reported other. In computation of the inverse probability weights (described below) we also used sampling strata as a covariate.mice R package (version 3.13.0) with five iterations to create each of five imputed datasets. For each imputed dataset, we obtained estimates of To obtain estimates that were applicable to the target population represented by the HCHS/SOL, we applied inverse probability weighting (IPW) to respondents’ data. Weights were estimated as survey R package (version 4.0).All analyses, including computations of percentages and association analyses, accounted for the stratified sampling strategy and clustering of HCHS/SOL participants by applying functions from the Primary analyses of genetic test awareness used a composite measure, where individuals were assigned value of 1 (aware) if they reported awareness of any of the four types of genetic tests described and 0 (unaware) if they were not aware of any test and answered at least one item about genetic test awareness. Individuals with no data on any genetic test awareness items because of refusals and missing data were considered non-respondent and omitted from analyses . A similar approach was used to classify whether participants were interested in these genetic tests, as well as to classify whether they had ever been offered or used them . Participants who were classified as don’t know/not sure on items about being offered or using genetic were included in analyses as not being offered or using those tests.We identified demographic and healthcare-related factors associated with genetic testing awareness using logistic regression. We identified demographic and healthcare-related factors associated with perceived usefulness using Poisson regression, because of the highly skewed distribution of the responses. We used contrasts to compare awareness and perceived usefulness of genetic tests between extreme levels of variables associated with awareness and perceived usefulness.Because of the small numbers of participants reporting being offered or using genetic tests, we performed unadjusted analyses reporting overall numbers of individuals and percentages for these outcomes, without performing association analysis. Findings were reported as statistically significant if p values were less than 0.01.In secondary analyses, we compared the characteristics of participants who participated in the AFU but did not respond to the genetic testing survey to those who responded , and repeated (fully adjusted) regression analyses of genetic testing awareness and perceived usefulness stratified by gender and by HCHS/SOL study center. Also, in supplemental analyses, we repeated the main analyses stratified by study center, because there could be systematic differences between regions with respect to genetic testing.Of 16,415 HCHS/SOL visit 1 participants, 9,408 participated in the study’s 2019–2020 AFU phone survey and 5,769 completed at least one item about genetic testing for an overall participation rate of 35.1% (unweighted), or 32.6% of the target population (weighted estimate). Individuals who participated in the AFU but did not complete any genetic testing awareness items tended to be less educated, had lower household incomes, were more likely to be single and retired, and were more likely to be enrolled at the Bronx site and less likely to be enrolled at the Chicago and San Diego sites than individuals who completed at least one awareness item  . CharactOverall, 2,891 survey responders, or 55.3% (weighted percentage) of individuals in the target population reported awareness of at least one of the four types of genetic tests. These included tests to determine risks for particular diseases, tests to determine the likelihood of passing inherited risks to children, tests to inform disease treatment, and tests to determine medication selection. Individuals were most likely to report awareness of genetic tests to determine risks of getting disease  and the likelihood of passing disease to children , and least likely to know about genetic tests providing information about how diseases should be treated  or determining medication selection . Few individuals reported ever being offered or receiving the types of genetic tests described in the survey . Only 36Few demographic or medical care factors that were examined by logistic regression analyses were associated with overall awareness of genetic testing . Some ofStratified analyses showed that some of the above-mentioned patterns were stronger at specific sites . DiffereAnalyses about specific genetic tests showed similar trends . DiffereOverall, participants rated the usefulness of genetic testing for managing a person’s health 8.4 (standard error = 0.05), on average, on a 1  to 10 (extremely useful) scale. Few demographic factors were associated with usefulness scores in Poisson regression models . AnalyseWe leveraged one of the largest, population-based cohort studies of U.S. Hispanic and Latino individuals to provide novel insights about demographic factors associated with genetic testing awareness in this growing population. Our findings showed that approximately 55% of this population was aware of genetic tests for health. This proportion was only slightly higher than findings from the 2017 HINTS, where just under one-half of Hispanic and Latino respondents and 57% of respondents overall reported awareness of health-related genetic testing.,,,,Our findings highlight concerns about the potential for important segments of Hispanic and Latino populations to miss opportunities to benefit from improvements in genetic testing. Tremendous advances have occurred in medical genetics in recent years. These include the refinement of approaches to assess genetic risk, such as polygenic risk predictions,,,,A number of differences observed in prior work were not observed in our analyses. Specifically, differences in Hispanic and Latino populations by gender, Hispanic or Latino background, and birth location reported from the 2005 HINTS data were not statistically significant in our analyses. Only a few demographic factors were associated with genetic testing awareness in our study population. Specifically, we observed differences in awareness between centers of enrollment and by income and education. A greater awareness of genetic testing among individuals with higher educational status and income was consistent with previous findings.Our study also showed that very few Hispanic and Latino participants remembered being offered or receiving genetic testing. HINTS data across populations from 2017 showed that 12% of respondents overall reporting ever having undergone a genetic test.,,,,,,,,Our work also showed relatively high perceptions among Hispanic and Latino participants about the usefulness of genetic testing. Interestingly, we observed greater perceived usefulness of genetic testing among individuals who were not U.S. born, which is contrasts with prior work with Hispanic and Latinos populations, which has found a lower awareness among individuals of lower acculturation and those who have resided in the United States for shorter times.The strengths of our study include the large sample size of an understudied population and post hoc adjustment to provide population-based estimates. Our analytic sample not only focused on Hispanics and Latino individuals, but also included large numbers of respondents with lower educational attainment and income. Limitations to our analyses included moderate completion rates for the genetic awareness questions and the use of brief questions that provide limited insight about the barriers to awareness and test use or why participants felt genetic testing was useful or not useful. We did not study psychosocial and cultural factors, such as religiosity, medical distrust, and acculturation, which may vary within the Hispanic and Latino population and may be associated with outcomes of interest, which remains a topic of future work. Participants were enrolled at four centers with limited representation from rural areas. Our descriptions of genetic tests were adapted from items used previously in the national HINTS instrument and do not reflect the range of health-related applications that currently exist. Furthermore, response rates to the HCHS/SOL AFU survey were moderate, and numerous individuals who started the survey did not provide data for the genetic testing section. Don’t know responses were analyzed as no throughout, and the findings may underestimate the proportion of respondents with genetic test awareness, as well as the proportion of respondents who had been offered and used genetic tests.Nevertheless, our work adds weight to evidence that use of genetic testing is limited in Hispanic and Latino individuals, although attitudes toward these applications are largely positive. These findings highlight the need to tailor genetic testing education and outreach to account for the wide variation in perception, needs, and potentially, believes, among Hispanic and Latino individuals. Education and outreach that accounts for the large diversity of Hispanic and Latino communities are likely to yield the greatest improvements in genetic test awareness and better ensure all individuals are able to capitalize on improvements in genomic medicine.https://sites.cscc.unc.edu/hchs/ for study procedures. HCHS/SOL data are also available on the National Heart Lung and Blood Institute’s BioLINCC (Biologic Specimen and Data Repository Information Coordinating Center) repository under accession number HLB01141422a.HCHS/SOL data are available via a data use agreement with the HCHS/SOL Data Coordinating Center. See https://github.com/tamartsi/Genetic_testing_awareness_SOL.Code is publicly available on the GitHub repository: The HCHS/SOL was approved by the institutional review boards at each field center, where all participants gave written informed consent during their clinical exam, and by the Non-Biomedical IRB at the University of North Carolina at Chapel Hill, to the HCHS/SOL Data Coordinating Center. The informed consent form included the statement “I (agree/do not agree) to allow HCHS/SOL staff to contact me once a year to ask questions about my health and where I live.”


Saccharomyces. Pediococcus, Companilactobacillus, and Weissella were the dominant bacterial genera in CTS and fermented dough. Adding alkali could retain the types of dominant yeasts and LAB derived from CTS, decrease the relative abundance of Companilactobacillus crustorum and Weissella cibaria, and increase that of Pediococcus pentosaceus, in fermented dough. Principal component analysis (PCA) indicated that adding alkali decreased the content of sourness-related volatiles in CSB fermented by CTS. Correlation analysis showed that Pediococcus and Weissella in fermented dough were positively correlated with the lipid oxidation flavor-related compounds in CSB, and Lactobacillus was positively correlated with sourness-related aroma compounds. Synthetic microbial community experiments indicated that CSB fermented by the starter containing P. pentosaceus possessed a strong aroma, and adding alkali weakened the flavor intensity. Alkali addition could promote the formation of ethyl acetate and methyl acetate with a pleasant fruity aroma in W. cibaria-associated CSB.Alkali is an indispensable additive in Chinese steamed bread (CSB) production. This work aimed to evaluate the key roles of alkali in the microbial community of dough fermented using Chinese traditional starter (CTS) and the aroma profiles of CSB. The dominant fungi in CTS and fermented dough were members of the phylum Ascomycota and the genus  Chinese steamed bread (CSB) is a fermented wheat flour food, a traditional staple food in northern China with >1500 years of history . General2CO3, provided a reliable solution [2CO3 could improve the flavor of CSB by inhibiting aroma-negative compounds [Accurate temperature, suitable acidity, and appropriate moisture are basic requirements for dough fermentation. Thus, CSB is usually made by experienced homemakers or workers. Historically, CSB makers used vegetation greywater or trona to neutralize the acidity produced during dough fermentation, thereby decreasing the unpleasant sourness of CSB. Because the quality of these ancient neutralizers was uncertain, the quality of the CSB product was also unstable. Fortunately, the advent of industrial food-grade alkali, that is, Nasolution . It rapisolution , corn tosolution , and presolution . Alkali solution , and Na2ompounds .Saccharomyces cerevisiae, Kazachstania humilis, Pichia kudriavzevii, and so on [Fructilactobacillus sanfranciscensis, Pediococcus pentosaceus, Companilactobacillus crustorum, Weissella cibaria, Lactiplantibacillus plantarum, etc. [Pediococcus was the predominant genus in CTS by high-throughput sequencing, and there were far more flavor compounds in CSB compared with commercial yeast-based CSB. Hu et al. [L. plantarum and S. cerevisiae cultures could replace naturally fermented doughs and improve the specific volume, texture, and aroma of bread. The combination of high-throughput sequencing and synthetic microbial community techniques could provide convenience for the study on the relationship between the microorganisms in CTSs and the aroma profiles of CSBs.CTSs are complex and stable biological ecosystems, containing bacteria, yeasts, and molds. The microbial composition and main strains have been the subject of many studies. The yeasts possess superior gas generation capacity and make CSB spongy and fluffy. The reported relevant yeast species include nd so on ,11. The um, etc. ,12. CTS-um, etc. . Suo et um, etc.  reportedu et al.  used a sS. cerevisiae cultures and determined the content of aroma compounds of CSBs fermented by these starters. This study bridges gaps in current knowledge and provides novel insights into the mechanisms underlying the improvement of CSB flavor via alkali addition.However, systematic study of the effect of adding alkali on the microbial composition in dough fermented by CTS and on the aroma profile of CSB has been ignored. Therefore, we investigated CTSs sampled from different regions in northern China, analyzed the fungal and bacterial communities in CTSs and fermented doughs with alkali addition, and then detected the aroma compounds in the corresponding CSBs by gas chromatography-mass spectrometry (GC-MS). Finally, we prepared synthetic microbial community starters for dough fermentation using LAB combined with 2CO3; Bakerdream Co., Ltd., Weifang, China) was purchased from a local supermarket.Hand-made or workshop-manufactured CTSs were sampled from eight cities in northern China : Wuwei CAccording to the recommendations of each CTS maker, the optimized method of CTS dough fermentation was as follows: First, 15 g CTS and 0.45 g alkali were pre-dissolved in 100 g aseptic water at 30 °C for 10 min, added into 185 g wheat flour, and mixed in a dough mixing machine  for 20 min. Then, 60 g of each dough was sampled to make a well-proportioned hemispherical shape and put into a fermentation cabinet to proof at 35 °C and 80% relative humidity. The fermentation time referenced the method of Suo et al. [https://www.ncbi.nlm.nih.gov/genbank/, (accessed on 27 June 2022)). Strains were stored in 30% glycerin solution at −80 °C.Yeast and LAB from CTS were isolated and cultivated using Yeast Peptone Dextrose (YPD) and De-Man Rogosa Sharpe (MRS) medium , respectively. The strains of yeast and LAB were identified using homology alignment of conserved DNA sequences. DNA was extracted from single colonies using a fungal or bacterial DNA extraction kit . PCR was used to amplify the internal transcribed spacer (ITS) rRNA gene of yeast and the 16S rRNA gene of LAB. The universal primer pairs used were ITS1/ITS4 and 27F/1492R, respectively. The program of amplification referred to the Kit instructions . The conditions were denaturation at 95 °C for 3 min; 35 cycles of 95 °C for 15 s, 58 °C for 30 s, elongation at 72 °C for 90 s; and a final extension at 72 °C for 5 min. The total reaction volume was a 20 μL mixture containing 10 μL of 2 × Phanta Max Buffer, 0.4 μL of 10 mM dNTP Mix, 0.8 μL of forward primer (10 μM) and reverse primer (10 μM), 0.4 μL of Phanta Max Super-Fidelity DNA Polymerase, and 1 μL of template DNA. The PCR products were sequenced by Wuhan AuGCT Biotechnology Co., Ltd. The sequencing data were submitted to GenBank , and they were mixed in a dough mixing machine for 20 min. Then, 60 g of each dough was sampled to make a well-proportioned hemispherical shape and put into a fermentation cabinet to proof at 35 °C and 80% relative humidity. When the proofing process was completed, the volume of the dough had doubled, and the inside was a dense honeycomb structure, with slightly sour flavor. Finally, the fermented dough was steamed above boiling water in a pot for 25 min, then left to rest at room temperature for 10 min to obtain CSB sample. Details of the strains are given in The yeast and LAB strains were cultivated in the corresponding liquid medium until the beginning of the stationary growth phase. Cells were collected from these cultures by centrifugation  at 20 °C and washed twice with aseptic distilled water. Collected cells were used immediately to make CSBs. CSBs fermented by yeast and LAB were manufactured as follows: Firstly, collected cells were directly added into 100 g aseptic distilled water and 200 g wheat flour (the added concentrations of yeast and LAB cells were 5 × 10Unfermented dough (20 g) was put into a graduated cylinder. The proofing conditions were the same as those used for preparation of CSB. The final volume was recorded. The specific volume was the ratio of the final volume to the initial weight.The pH and TTA values were measured according to the previously described method of Yan et al. . Ten graTotal DNA was extracted from fermented dough using a total DNA extraction kit (Omega Inc.). The hypervariable regions of fungi 18S rRNA gene were amplified with primers SSU0817F (5′-TTAGCATGGAATAATRRAATAGGA-3′) and 1196R (5′-TCTGGACCTGGTGAGTTTCC-3′). For bacteria, the V3 and V4 hypervariable regions of 16S rRNA were amplified using primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). Purified amplicons were pooled in equimolar amounts, and paired-end sequencing was performed on Illumina MiSeq PE300 platform/NovaSeq PE250 platform according to standard protocols. Operational taxonomic units (OTUs) with 97% similarity cutoff were clustered using UPARSE version 7.1.m/z.According to the GC-MS method of Xi et al. , the volVolatile compounds were identified individually by matching mass spectra with the NIST20 library database . Ethyl hexanoate  was added into samples as an internal standard for semi-quantitative calculation.p < 0.05.GraphPad Prism 8  software was used for statistical analysis, with differences being considered statistically significant at 2CO3 could significantly increase the specific volume of buckwheat CSB to 2.07 and 2.16 mL/g, respectively, by neutralizing the organic acids.The leavening capacity of starter is the most important factor that affects the quality of steamed bread. The specific volumes, pH, and TTA values of fermented doughs were determined and are shown in The pH values of AFDs ranged from 4.47 to 6.22, and the TTA values were from 1.6 to 3.3. For FDs, the pH values ranged from 4.16 to 5.44, and the TTA values were from 2.0 to 5.6. The pH and TTA values differed among the doughs fermented by CTSs from different regions. The acidity of fermented dough was mainly related to the microbiota in the starters .2CO3 dissolution, which promotes the generation of CO2, and provides a suitable environment for microorganisms to maintain high gas-producing capacities [Overall, the specific volume and acidity of fermented doughs varied in different regions. The addition of alkali could increase specific volume and decrease acidity in fermented dough. These findings can be explained in two ways. First, the complex origin of raw materials and the various production processes of CTSs bring different microorganisms into CTSs, leading to different fermentation properties of doughs, including gas production and acid accumulation . Second,pacities ,17.High-throughput sequencing of 18S rRNA gene was performed to analyze the fungal communities in CTSs and fermented doughs. Sequencing quality, community diversity, and community richness were evaluated by the rarefaction curve, the Shannon index, and the Chao1 index, respectively. The sequencing and diversity results of fungi are shown in Saccharomyces, Aspergillus, Hyphopichia, Fusarium, and Pichia. Consistent with previous research, Ascomycota was the predominant phylum in CTSs, Saccharomyces was the dominant genus in the CTS group (98.1%), and their relative abundance increased further in the AFD group [S. cerevisiae, Aspergillus penicillioides, Hyphopichia burtonii, Fusarium graminearum, and P. kudriavzevii  and YC (6.7%) CTSs, and it was reported to be the predominant fungus in sufu, a traditional Chinese appetizer of fermented soybean curd [H. burtonii causes “chalk mold” on bread [F. graminearum is a pathogenic fungus that causes wheat scabs [As shown in FD group . The funiavzevii B. S. cerparation . P. kudrosystems . A. peniean curd . H. burton bread , and F. at scabs ; even thBacterial communities were analyzed by high-throughput sequencing of 16S rRNA gene. The sequencing and diversity results for bacterial communities are shown in Pediococcus, Companilactobacillus, Weissella, Fructilactobacillus, Levilactobacillus, Latilactobacillus, Acetobacter, Limosilactobacillus, and Gluconobacter [Pediococcus, Companilactobacillus, Weissella, Fructilactobacillus, Levilactobacillus, Latilactobacillus, and Limosilactobacillus were common LAB and always found in fermented food. The predominant genus of bacteria in CTS group was Pediococcus, and its relative abundance was higher in the FD and AFD groups than in the CTS group, revealing that Pediococcus was enriched during dough fermentation. Adding alkali increased the relative abundance of Pediococcus from 37.8% in the FD group to 41.1% in the AFD group. Companilactobacillus was the sub-abundant genus in the CTS group, and adding alkali decreased its relative abundance in the AFD group. The third abundant genus was Weissella in the CTS group. Notably, Acetobacter and Gluconobacter were detected in the CTS group, but almost none of them was observed in the AFD and FD groups. This could be explained by the following reason: Acetobacter and Gluconobacter were originated from the raw materials  of CTSs, and they could not survive in dough. DNA from dead cells of Acetobacter and Gluconobacter in CTSs were detected by high-throughput sequencing. In the fermentation process, addition of wheat flour and water led this DNA to greatly diluting, and DNA was also degraded during the leavening process [The phylum Firmicutes was dominant , with renobacter ,24. Amon process .P. pentosaceus, C. crustorum, Pediococcus acidilactici, W. cibaria, F. sanfranciscensis, Levilactobacillus brevis, Latilactobacillus curvatus, Limosilactobacillus pontis, Gluconobacter oxydans, and Gluconobacter frateurii. P. pentosaceus and C. crustorum are usually found in fermented wheat flour products and demonstrated to be homofermentative LAB in sourdough, contributing significantly to the development of steamed bread flavor [P. pentosaceus was an efficient starter that improved bread quality and flavor by forming specific organic acids and volatile compounds. P. pentosaceus was present in the samples from LY, LK, HZ, and WH and predominated in LK and WH samples. C. crustorum was the most abundant species in WN samples; its relative abundance was decreased by adding alkali in fermented doughs. Comasio et al. [C. crustorum could convert the citrate into L-lactic acid, acetoin, and diacetyl in sourdough and endow wheat bread with a buttery odor. In addition, W. cibaria and F. sanfranciscensis, which are also obligate heterofermentative LAB [W. cibaria increased from 62.8% in CTS to 98.3% in FD from BZ, indicating that W. cibaria was highly competitive in this dough ecosystem. F. sanfranciscensis was a typical LAB in European type Ⅰ sourdough and the predominant species in CTS, AFD, and FD from YC, with relative abundances of >96.5%. L. pontis showed poor competitiveness in dough fermentation, with relative abundance in LK samples decreasing from 25.9% in CTS to 1.2% in FD.A Venn plot was used to analyze the similarity and specificity of bacterial species among the CTS, FD, and AFD groups A, indicad flavor . For exad flavor  clarifieo et al.  reportedtive LAB , dominatP. pentosaceus, P. acidilactici, F. sanfranciscensis, and L. brevis increased, while that of C. crustorum, W. cibaria, and L. curvatus declined in the AFD group compared with the FD group  was the most concentrated acid, with a concentration of 21.26 mg/kg in CSB from WN, which dropped to 6.07 mg/kg in the corresponding alkali-added CSB. Pentanoic acid (sweaty), hexanoic acid (sweaty), and heptanoic acid (cheese) were identified in all alkali-free CSBs and were decreased or even undetectable in alkali-added CSBs. Similar results were reported by Xi et al. , who obs2CO3 addition could significantly increase the concentration of hexanal in sourdough CSB. In addition, ethyl acetate (fruity) and 2-pentylfuran (fruity) were found in all the samples, and these volatiles also imparted CSB typical aromas [Aldehydes including hexanal (green), heptanal (fatty), octanal (citrus), (Z)-2-heptenal (green), nonanal (citrus), benzaldehyde , (Z)-2-nonenal (fatty), phenylacetaldehyde (honey-like), -2,4-nonadienal (deep fat fried), and -2,4-decadienal (deep fat fried) were detected in all CSBs. Hexanal was the most abundant aldehyde and was more abundant in alkali-added CSB than in alkali-free CSB from most regions, with the highest concentration in CSB from WH, at 26.78 mg/kg. This was essentially consistent with the study by Xi et al. , which sl aromas .PCA was performed to analyze the effect of adding alkali on the aroma compounds in CSB A. The fiLactobacillus  [Pediococcus, and Weissella in fermented dough, and the results are shown in To illustrate the relationships between aroma profiles of CSBs and bacteria in the corresponding fermented dough, correlation analysis was performed on the content of aroma compounds in CSBs and the relative abundance of three dominant genera including s, etc.) , PediocoZ)-2-heptenal, nonanal, and -2,4-nonadienal) and ketones , and four kinds of alcohols . Moreover, almost all of the short-chain aldehydes (C6–C9) were distributed in this cluster, and they are generally believed to be formed by auto-oxidation of unsaturated fatty acids from wheat flour, with hexanal mainly generated from C18:2, and the others from C18:1 [Pediococcus and Weissella in fermented dough were positively correlated with short-chain aldehydes, while Lactobacillus was negatively correlated with them, suggesting that dough fermented by Pediococcus or Weissella had more lipid-oxidation products than the dough fermented by Lactobacillus. Similar results were found by Xu et al. [P. pentosaceus participated in the lipid oxidation of broccoli juice fermentation, affected the content of lipid degradation products including volatile aldehydes and alcohols, and enhanced the sensory properties of broccoli juice. Lipid oxidation in wheat flour and microbial metabolism are the main pathways for the formation of alcohols in fermented dough. In the lipid oxidation pathway, the alcohols are generated by lipoxidase-catalyzed oxidation of fatty acids and conversion of lipid-oxidizing compounds such as aldehydes to the corresponding alcohols [Pediococcus and Weissella in fermented dough were positively correlated with 1-hexanol (green), 1-nonanol (citrus), and 1-heptanol (green), but Lactobacillus was negatively correlated with them. It was indicated that the fermented doughs containing Pediococcus and Weissella showed stronger lipid oxidation reactions, which provided more precursors for alcohol conversion and formed more alcohol flavor compounds than Lactobacillus in dough. Similarly, ketones, which are also characteristic flavor compounds in wheat bread, were principally produced via lipid oxidation [The aroma compounds of CSBs were divided into two clusters using hierarchical clustering. The first cluster included most of the aldehydes , in dough co-fermented by LAB and yeast [Almost all of the acids, including acetic acid (sour), propanoic acid (rancid), butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, and octanoic acid (cheese), were classified in the second cluster. eissella . Secondlnd yeast .P. pentosaceus, C. crustorum, and W. cibaria were combined with S. cerevisiae as synthetic microbial community starters, respectively. The contents of aroma compounds in CSBs are shown in To further analyze the effects of different dominant LAB and alkali addition on the aroma profiles of CSB, referring to the microbial community analysis described above, P. pentosaceus were more abundant than those in CSB fermented by C. crustorum or W. cibaria, such as phenylethyl alcohol, 3-methyl-1-butanol , and ethanol. Phenylethyl alcohol and 3-methyl-1-butanol are typical higher alcohols in fermented dough. Higher alcohols are converted by the corresponding branched-chain amino acids via Ehrlich pathway in an anaerobic environment; for example, phenylethyl alcohol is generated from phenylalanine [S. cerevisiae metabolism under anaerobic conditions [P. pentosaceus had a strong aroma with high concentrations of Ehrlich volatiles and ethanol, which indicated that P. pentosaceus might be better at promoting Ehrlich and EMP pathways in S. cerevisiae metabolism than C. crustorum and W. cibaria, in the dough ecosystem. With the addition of alkali, the content of 17 compounds such as pentanoic acid, hexanoic acid, heptanoic acid, hexanal, octanal, 1-hexanol, and 1-octen-3-ol significantly decreased in CSB, suggesting that adding alkali could weaken the aroma profile of CSB fermented by P. pentosaceus.Notably, >50% of the aroma compounds in CSB fermented by lalanine . Ethanolnditions . We founW. cibaria and P. pentosaceus, and only pentanoic acid displayed a significant decrease in CSB fermented by C. crustorum, which might be related to the higher tolerance of C. crustorum to alkali than W. cibaria and P. pentosaceus in the dough ecosystem. Acetic acid has been demonstrated to impact the aroma of wheat flour bread negatively at high concentrations [W. cibaria was the highest (average 15.92 mg/kg), >1.5-fold that in C. crustorum-fermented CSB (average 9.45 mg/kg). The heterofermentative fermentation property of W. cibaria might be responsible for the accumulation of acetic acid in the fermented dough [W. cibaria directly converts glucose to lactic acid and acetic acid via the pyruvate metabolism pathway, but lactic acid is the only byproduct of glucose in homofermentative C. crustorum, in the dough ecosystem. Heterofermentation can also produce esters that impart a pleasant and fruity odor in bread [W. cibaria. Notably, adding alkali significantly decreased (by >2-fold) acetic acid in W. cibaria-fermented CSB, and slightly decreased that in CSB fermented by P. pentosaceus or C. crustorum. Thus, it was indicated that heterofermentative W. cibaria accumulated more acetic acid than P. pentosaceus or C. crustorum in fermented dough, and adding alkali not only neutralized acetic acid but also promoted the formation of ethyl acetate and methyl acetate in CSB.Similarly to the results of aroma compound analysis of CSB fermented by CTS, the addition of alkali decreased almost all of the volatile acids in CSB fermented by synthetic microbial community starter. Pentanoic acid, hexanoic acid, and heptanoic acid showed significant decreases in CSBs fermented by trations . The coned dough .HeterofePediococcus, Companilactobacillus, and Weissella. Adding alkali did not change the types of dominant LAB derived from CTS but changed their relative abundance in fermented dough. Adding alkali decreased the concentration of volatile acids in CSB. Pediococcus and Weissella were positively correlated with lipid oxidation flavor-related compounds in CSBs; Lactobacillus was positively correlated with sourness volatiles, suggesting that Lactobacillus had strong acid-producing capacity. The synthetic microbial community experiment exhibited that CSB fermented by P. pentosaceus had a strong aroma, and adding alkali weakened the flavor. CSB fermented by heterofermentative W. cibaria contained abundant acetic acid, and adding alkali decreased its concentration, promoted the formation of esters, and endowed CSB with a pleasant fruity aroma. Overall, the addition of alkali not only neutralizes organic acid in fermented dough, but also affects microbial metabolism, thereby reducing sourness and changing the lipid oxidation flavor and ester aroma of CSB. This study illustrates the mechanism underlying the improvement in the flavor of CSB via alkali addition and will provide a theoretical foundation for quality improvement and industrial production of steamed bread.To our knowledge, this is the first systematic study to investigate the effects of adding alkali on the microbial community in CTSs and the aroma profiles of CSBs, revealing the internal relationship between LAB and aroma characteristics of CSBs. The dominant bacteria at the genus level in CTS were