The use of skin toning products has a deep historical background in low and middle-income countries. Yet, there is no empirical evidence on the prevalence, and patterns of skin toning practices among university students in Ghana. This study sought to examine the prevalence, patterns and socio-demographic factors associated with skin toning practices among female university students in Ghana using a sample of 389 undergraduate female students.40.9% of respondents had practised skin toning within the last 12 months. Also, 51.3% used skin toning products such as creams (38.9%) and soap or gel (35.5%) to treat a skin disorder. Respondents aged 21 years were more likely to use skin toning products , those who had dark skin , attended public school  and those who attended girls school were more likely to use skin toning products . Furthermore, those who were in level 400  and those receiving more than 500 cedis were also more likely to use skin toning products . Policy interventions that seek to reduce skin toning practices among university students should consider micro and broader socio-demographic factors.The online version of this article (10.1186/s13104-019-4327-8) contains supplementary material, which is available to authorized users. Skin toning practice appears to have become a norm among people of various backgrounds, age, and gender –3. SeekiAlghamdi  reportedIn Africa, the World Health Organization claims that Nigeria has the highest percentage of women using skin toning products with reported 77% of women engaging in the practice . A crossSkin toning practices have been reported among young women in Cameroon , and amoA cross-sectional University-based survey was conducted at the Kwame Nkrumah University of Science and Technology (KNUST) to examine patterns and prevalence of skin toning practices among female university students in Ghana. Being the second largest university in Ghana, KNUST is located in Kumasi and provides educational services for several people in Ghana and other neighbouring countries. This study recruited female undergraduate students from levels 100 to 400. Female students from the various colleges of the university such as Humanities and Social Sciences, Arts and Built Environment, Science, Health Science and Agriculture, and Natural Resources were selected using a two-stage cluster and random sampling techniques. Out of the 13,738 female students at KNUST, a formula by Miller and Brewer  were useIn each college, the number of respondents was calculated proportionately using the population of the undergraduate females in the various colleges. The respondents were asked to pick pieces of papers that were folded with ‘True’ and ‘False’ options. Those who chose ‘True’ were selected until all the sample size earmarked for each college was obtained. The recruitment of the respondents for the study is shown in Fig. A closed-ended questionnaire  (SPSS) was employed to establish an association between socio-demographic characteristics of the respondents and the use of skin toning products with a significant level of 0.05 or less.Data gathered on the demographic characteristics of respondents were presented in Table On the prevalence and patterns of skin toning practices among female university students  used skin toning products to treat a skin disorder. Moreover, Creams (38.9%) and Soap or Gel (35.5%) were the skin toning products mostly used by the respondents.= 0.400, CI 0.121–1.320). Respondents who had dark skin were 3.3 times more likely to use skin toning products . Those who attended public school were 1.9 times more likely to use skin toning products  and those who attended girls school were 10.7 times more likely to use skin toning products . Furthermore, those who were in level 400 were 49 times more likely to use skin toning products  and those who received more than 500 cedis were 2 times more likely to use skin toning products  as shown in Table In the multivariate analysis, the results show that respondents aged 21 years were 0.4 times more likely to use skin toning products  had practiced skin toning within the last 12 months prior to the survey. Age, skin colour, nature of school attended, type of school attended, level of student and monthly income significantly influence the use of skin toning products among university students in Ghana. We, therefore, argue that policy interventions that seek to reduce skin toning practices among university students should consider micro and broader socio-demographic factors.The study was limited to the views of female university students; however, the inclusion of male university students could have paved the way for new findings. Further, the use of one institution and the period within which data was collected limits the extent to which the findings could be generalized. It is, therefore, recommended that future research should be extended to students in other universities and also consider the views of male students on the use of skin toning products.Additional file 1. Questionnaire.Additional file 2: Table S1. Prevalence and patterns of skin toning practices among female university students.Additional file 3: Table S2. Socio-demographic characteristics influencing the practice of skin toning.


Research of bacterial communities and metabolism potential of paddy soils contaminated by antimony (Sb) and arsenic (As) are vital to acquire understanding for their bioremediation. Here, the relative abundance of Sb and As metabolism genes, the diversity and composition of the bacterial community, and the influences of geochemical properties and the bacterial community and metabolism potential have been researched by Tax4Fun2 prediction and high-throughput sequencing. LEfSe  analysis shown different taxa were enriched in dissimilar soil layers. RDA  and relative importance analysis indicated the main properties including total sulfur (TS), total organic carbon (TOC), pH, and the bioavailable fractions of Sb and As affects the bacterial community, which Sbrec, Astot, and Asrec had greater impact on the bacterial taxonomic community. For example, Asrec, Astot, and Sbrec had a positive correlation with Chloroflexi and Rokubacteria, but negatively correlated with Proteobacteria and Actinobacteria. Obtaining metabolic function genes by using the tax prediction method. RDA, relative importance analysis, and co-occurrence network analysis showed the geochemical properties and bacterial community affected Sb and As related bacterial functions. The partial least squares path model (PLS-PM) analysis indicated Sb and As contamination fractions had negative effects on ecological function, bacterial community structure had positive influences on ecological function, and the direct effects of geochemical properties on ecological function was greater than community structure. The direct impact of As contamination fractions on bacterial community structure was greater than Sb, while the direct impact of Sb contamination fractions on bacterial function was more remarkable than As. Obviously, this study provides a scientific basis for the potential of biochemical remediation of Sb and As contamination in paddy soils profile. Due to ony Mine . The higony Mine ,4,5,6.Microorganisms are critical factors in trace elements migration, transformation, methylation, and remediation because of their bioavailability of trace elements. It was reported that Sb widely existed in the lithosphere and was liable to form symbiotic sulfides with As , and theThe relative abundance, diversity, and activity of genes related to Sb and As transformation often determine the migration and transformation of Sb and As. Reductive genes (arrA and arsC), which affect the reduction of As (V) and mediate respiratory and detoxification pathways, oxidase genes (aioA) and small subunits of oxidase (aoxA and aoxB) promote the oxidation of As (III), and transferase genes  mediate migration, transformation, and methylation pathways ,10. AlthIn this study, we selected the paddy soils adjacent to Banpo Antimony Mine and a smelter in Dushan County, southwest China. In order to understand the effect of Sb and As contamination fractions as well as other geochemical properties on the structure and function of bacterial community in paddy soil profile, our hypothesis was that the characteristics of the bacterial community and function in different soil layers and its responses to Sb and As contamination were different. To test the hypothesis that Sb and As contamination fractions in paddy soil, we measured the bacterial communities and functions by using a 16S rRNA  sequencing technique. The aims of this study are: (1) to understand the distribution of Sb and As contamination fractions as well as other geochemical properties in paddy soil profile; (2) to clarify the distribution characteristics of bacterial communities and functions in paddy soil profile; and (3) to determine the potential relationship between Sb and As contamination fractions as well as other geochemical properties and soil bacterial communities and functions.Sampling sites were located in the paddy soils around Banpo Antimony Mine and Xiaohe Antimony Mine Smelter in Dushan County, Guizhou Province, Southwest China. Banpo Antimony Mine has been in operation since the last century, and Xiaohe Antimony Mine Smelter has been smelting since 1970. Therefore, the accumulation of Sb and As has been present in the paddy soils around them. Five soil profiles with a width of 1 m and a depth of 1 m were collected in the paddy soils around the antimony mining area and the smelting area. Two soil samples of the profiles were collected from the Banpo antimony mining area, whereas three soil samples were collected from the smelting area. Moreover, the profile from the surface to the bottom was three layers: SL1 (0–30 cm), SL2 (30–65 cm), and SL3 (65–100 cm). Paddy soil was greatly affected by human activities, and according to the classification of soil taxonomy , the pad−1 of HCl for 30 min with shaking, and allowed to equilibrate for 4 h. The supernatant was then collected by centrifugation at 3500 rpm for 10 min. Finally, the total Fe concentration was determined by 1,10-phenanthroline spectrophotometry at 510 nm.All paddy soil samples were pre-treated after being naturally air-dried. Plant roots, leaves, and gravels form the paddy soil were ground and passed through 2 mm sieve. Then these soil samples were further thoroughly ground and passed through a 200-mesh sieve. Total sulfur, total organic carbon, and the carbon–nitrogen ratio were determined by an elemental analyzer ; and pH was used to determine the glass electrode method (water–soil ratio 2.5:1). Total Fe was determined as described previously ,23. Brie−1 of CH3COOH. Next, the reducible fraction was extracted using 40 mL 0.5 mol·L−1 of NH2OH·HCl. The oxidizable fraction was then extracted by 20 mL 1.0 mol·L−1 of CH3COONH4 after oxidization using 10 mL 8.8 mol·L−1 of H2O2.The supernatant was injected into a polyethylene container and stored in a refrigerator at 4 °C for testing. Then HG-AFS  was used to determine Sb and As contamination fractions. Duplicate samples (repeated three times for each sample), reagent blanks, and standard reference materials  were included during the procedure to ensure the analytical accuracy. The percentage difference of the duplicate samples was under ±5%, and the metal recoveries of Sb and As in the standard reference materials were 92% and 95%, respectively.The contamination fractions of Sb and As were determined by BCR continuous extraction method ,25. Brie® soil DNA Kit  according to the manufacturer’s protocols. The final DNA concentration and purification were determined by NanoDrop 2000 UV-vis spectrophotometer , and DNA quality was checked by 1% agarose gel electrophoresis. The V3–V4 hypervariable regions of the bacterial 16S rRNA gene were amplified with primers 338 F (5’-ACTCCTACGGGAGGCAGCAG-3’) and 806 R (5’-GGACTACHVGGGTWTCTAAT-3’) by the thermocycler PCR system . The PCR reactions were conducted using the following program: 3 min of denaturation at 95 °C, 27 cycles of 30 s at 95 °C, 30 s for annealing at 55 °C, and 45 s for elongation at 72 °C, and a final extension at 72 °C for 10 min. PCR reactions were performed in triplicate 20 μL mixture containing 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu polymerase, and 10 ng of template DNA. Microbial DNA was extracted from soil samples using the E.Z.N.A.The resulting PCR products were extracted from a 2% agarose gel and further purified using the AxyPrep DNA Gel Extraction Kit  and quantified using QuantiFluor™-ST  according to the manufacturer’s protocol. Purified amplicons were pooled in equimolar and paired-end sequenced (2 × 300) on an Illumina MiSeq platform  according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. .Soil bacteria 16S rRNA data processing: The original sequencing sequence was spliced by QIIME2  and DADAThe environmental factors and bacterial community structure were analyzed by PCoA  and RDA using the ape package , vegan p−1 in Sbtot, and from 30.84 to 92.75 mg·kg−1 in Astot, respectively. In the bioaccessible fractions of Sb, the exchangeable fraction (Sbaec) and the oxidizable fraction (Sboxz) increased with the soil depth increase, and the reducible fraction (Sbrec) remained unchanged with an increase of soil depth. In the bioaccessible fractions of As, the concentration of fractions remained the same as the soil layers increased. Other geochemical properties of the various layers of the paddy soil profile were compared  was established to explore the direct or indirect relationship between soil bacterial function, soil physicochemical properties, and bacterial community structure D. Soil g2 and Fe (OH)3 formed by soil had strong adsorption capacity for Sbrec, so that the content of Sbrec was still high in SL2 and SL3. The concentration of Sboxz increased with the depth of soil layer, which might be due to the accumulation and transformation of other contaminated fractions of Sb by microorganisms. With the increase of soil depth, the content of Sbtot decreased gradually, while the As contamination fractions remained basically unchanged with the increase of soil layer. Since the chemical properties of Sb and As were similar, it could be seen that the sources of Sb and As might be different in paddy soil profiles. Sb contamination might come from atmospheric deposition or artificial release, while As contamination might come from parent rock, which was similar to previous research results [Even though there were relatively more studies on antimony mining waste and surrounding soil, sediment, and water environmental media ,38,39,40 results .−1 and had little effect on the composition of bacterial community [−1. PCoA analysis showed that there were significant differences between SL1 and SL2, SL1 and SL3 in the bacterial community composition in different layers of paddy soil, and the similarities between SL2 and SL3 were higher. LEfSe analysis further suggested that different taxa were enriched in different soil layers, which reflected the characteristics of the soil layer to some extent, which was similar to previous research results [Trace elements contamination affected the community structure, richness, and diversity of bacteria in environmental media. High concentrations tended to reduce bacterial abundance and diversity ,51, whilommunity . Our res results . The ext results . Previou results ,56. In o results ,58, and  results . We founThere are some bacterial taxa with multiple functions in different ecosystems . In thisAnaeromyxobacter belongs to Proteobacteria, which was a typical As-reducing bacteria. It was found that As was detected mostly in contaminated soil. It has the function of metabolizing various electron receptors and can reduce As (V) [Anaeromyxobacter mainly existed in SL3, and increased with the deepening of the soil layer. Through symbiotic network analysis, we found that Anaeromyxobacter (genus11) had a negative correlation with arsenate reductase , which further confirmed that Astot has a negative impact on Proteobacteria. Geobacter [Sphingomonas [Sphingomonas had a positive response to As contamination fractions but had no response to Sb contamination fractions. It was further showed that Proteobacteria was apparently greatly affected by the As contamination fractions during the migration and transformation of Sb and As.Co-occurrence network analysis showed that genus1 of Anaerolineaceae was positively correlated with As-related genes such as arsC1, arsC2, arsA, and arsR, indicating that it might participate in the reduction and migration transformation process of Sb and As in the biochemical cycle, which further confirmed that Asrec had a tremendous positive impact on Chloroflexi. e As (V) . Anaeromeobacter , and Sphngomonas  were typStaphylococcus xylosus plasmid pSX267 to Sb (III) and As (III) resistance decrease [The prediction of Tax4Fun2 indicated that microorganisms mediated the redox reaction of As in soil layers. Due to the similar chemical structure of Sb and As, soil microorganisms may migrate and transform Sb and As through similar metabolic pathways. The relative abundance of arsR, arsC2, and ACR3 in different soil layers was higher and decreased with the increase of soil layers. The reason was that the resistance genes to Sb contamination were weakened by the decreased concentration of Sb. It was reported that inactivation of arsR results in decrease . RDA anadecrease .Agrobacterium tumefaciens can oxidize Sb (III) [Comamonas, while Meng et al. (2004), found that arsB could catalyze the transport of Sb (III) [Similarly, the oxidation of Sboxz may also contribute to the enrichment of As oxidase (aoxA and aoxB) or the presence of oxidase-like enzymes. Previous reports have found that Sb (III) , althougSb (III) , existedSb (III) . These rThrough the above RDA analysis and relative importance analysis, we found that both Sb and As contamination fractions and other geochemical properties affected the bacterial community structure and function. In order to further understand how bacterial function was affected by biological and abiotic factors in paddy soils, PLS-PM was used to describe the direct and indirect relationships between different variables. The results show that the direct impact of As contamination fractions on bacterial community structure was greater than that of Sb, which was consistent with previous study . The dirIn this paper, we quantified the effects of Sb and As contamination fractions and other geochemical properties on bacterial community and function in the vertical profile of paddy soils. Soil layer increased with decreasing Sb contamination fractions, while the As contamination fractions remained basically unchanged. The difference between Sb and As contamination fractions in different soil layers made different taxa of the bacterial community enrich in different soil layers. This enrichment might change with the vary of Sb and As contamination fractions. The main factors affecting the structure of bacterial community were TS, TOC, and pH as well as the contamination fractions of Sb and As, and the stability of soil bacteria. It was followed by SL1 > SL2 > SL3 when facing environmental changes such as Sb and As contamination in different soil layers. In addition, the direct influence of Sb and As contamination fractions on bacterial function was more significant than that of bacterial community structure. The direct impact of As contamination fractions on bacterial community structure was higher than Sb, while the direct impact of Sb on bacterial function was higher than As contamination fractions. The focus of the effects of Sb and As contamination on bacterial community and function provided knowledge for the geochemical process and biotransformation of Sb and As, which allows the development of strategies for the biochemical remediation of Sb and As contamination in the vertical profile of paddy soils.


Compared to the control meal (plain rice + plain water), the addition of test extracts in either form did not modulate PPG, PPI, or BP. However, the extracts when added within rice while cooking gave rise to significantly lower PPI than when consumed as a drink (p < 0.01). Therefore, the form of consumption of phytochemical-rich ingredients can differentially modulate glucose homeostasis. This study also highlights the need for undertaking randomized controlled clinical trials with traditional foods/components before claims are made on their specific health effects.Several plant-based traditional ingredients in Asia are anecdotally used for preventing and/or treating type 2 diabetes. We investigated three such widely consumed ingredients, namely corn silk (CS), cumin (CU), and tamarind (TA). The aim of the study was to determine the effects of aqueous extracts of these ingredients consumed either as a drink (D) with high-glycemic-index rice or added to the same amount of rice during cooking (R) on postprandial glycemia (PPG), insulinemia (PPI), and blood pressure (BP), over a 3 h measurement period. Eighteen healthy Chinese men (aged 37.5 ± 12.5 years, BMI 21.8 ± 1.67 kg/m Arising from traditional dietary and alternative medicine practices across Asia  there have been a plethora of food-based components claimed as beneficial in improving the risk of diabetes and other cardiometabolic conditions. Unfortunately, the majority of these traditional practices have been based on historical pretext rather than on solid scientific evidence. In fact, robustly controlled clinical trials have been sparse, and most of the research claims came from in vitro studies and/or in vivo animal studies. In general, these traditional ingredients are all plant-based ingredients, rich in polyphenols and other phytonutrients with a wide range of biological functions including α-amylase and α-glucosidase enzyme inhibition, anti-inflammatory and antioxidant properties, insulin secretagogues, etc. ,2,3. In Stigma maydis) is a waste by-product of corn and has been long used in traditional Chinese medicine to treat diabetes and associated conditions [Corn silk ” study, the primary aims were to assess the three ingredients, viz., corn silk, cumin, and tamarind as listed above, in terms of their effects on postprandial glycemia and insulinemia, when consumed with a carbohydrate-rich meal principally consisting of a high-glycemic-index (GI) white rice. The secondary aim was to also measure the effects of these three ingredients on postprandial blood pressure. The study used aqueous extracts which can be obtained using household preparation methods. For cumin and tamarind, we used dietary doses, whereas for corn silk we used two separate doses—the “high dose” consisting of an amount commonly used in traditional Chinese medicine, and a lower  dose to assess whether this lower, “dietary” dose would still be effective in improving glycemic parameters. Moreover, there is now considerable evidence that the food form plays an important role in determining glycemic and/or insulinemic responses ,22,23. W2, waist circumference ≤90 cm, fasting blood glucose <7.0 mmol/L, blood pressure <140 mmHg and <90 mmHg for systolic and diastolic, respectively. Interested volunteers attended a screening visit after an overnight fast (no food or drink except water for a minimum of 10 h). At the screening visit, height was measured using a stadiometer , weight and body fat percentage were measured using the Tanita analyzer , seated resting blood pressure was measured using an automated sphygmomanometer . Fasting glucose was measured in whole blood using a HemoCue 201 RT analyzer  upon obtaining capillary blood using a single-use lancet device . Volunteers were excluded if they met any one of the following criteria: taken part in sports at the competitive and/or endurance levels; smoking; being allergic to common food  and test ingredients; intentionally restricting food intake; have glucose-6-phosphate deficiency (G6PD) or metabolic diseases, cardiovascular diseases, or disorders involving the gut, liver, or kidney; taking prescribed medication or dietary supplement that affects glycemia or interferes with other study measurements; consume ≥6 alcoholic drinks per week; donated blood within 4 weeks of study participation; had poor veins impeding venous access; had a history of severe vasovagal syncope following blood draws; had or was a carrier of a chronic infection ; suffering from active tuberculosis (TB) or receiving treatment for TB; or was related to a member of the study team. All volunteers provided written informed consent. All volunteers were given a choice of attending five or nine test sessions. This study was reviewed and approved by the National Healthcare Group (NHG) Domain-Specific Review Board, Singapore (DRSB Ref: 2018/00258). The study has been registered on clinicaltrials.gov under study ID No. NCT03685916. Eighteen volunteers were enrolled in this study through flyers placed around The National University of Singapore and from our center’s recruitment database of volunteers who had previously given consent to be contacted for research. Inclusion criteria for this study were: Chinese, male, age between 21 to 60 years old, body mass index (BMI) between 18.5 and 25.0 kg/mDried, mature corn silk, commonly sold as an ingredient to treat diabetes by traditional Chinese medicine (TCM) practitioners, was purchased locally. Aqueous extracts were obtained by adding 552 g of corn silk to 2.3 L of tap water and boiling for 15 min. A total of three batches of extraction were undertaken. After pooling, the total volume of the corn silk extract (CS) was made up to 6.9 L and filtered through a fine mesh strainer to remove corn silk particles. Then, 50 mL of this concentrated extract (24 g/100 mL) was aliquoted and stored frozen in a −20 °C freezer until further use. An aqueous extract of cumin was prepared by adding 552 g of ground cumin powder  Pvt. Ltd., Tamin Nadu, India) into 2.4 L of tap water and allowing the mixture to boil for 15 min. A further 2.4 L of tap water was added to the viscous mixture and boiled for an additional 15 min. Finally, the cumin extract (CU) was filtered through a fine mesh filter and the volume made up to 2.3 L and filtered through a fine mesh strainer to remove cumin particles. Then, 50 mL of this concentrated extract (24 g/100 mL) was aliquoted and stored frozen in a −20 °C freezer until further use. For the tamarind extract (TAM), 460 g of deseeded tamarind pulp  was added to 1.2 L of tap water and boiled for 15 min. The first aqueous extract was obtained by filtering the mixture through a fine mesh filter and the residual tamarind pulp re-extracted with another 1.2 L of tap water via boiling for another 15 min. The mixture from the second extraction was filtered and both tamarind aqueous extracts were pooled and the volume made up to 2.3 L. Then, 50 mL of this concentrated extract (20 g/100 mL) was aliquoted and stored frozen in a −20 °C freezer until further use. All stored concentrated extracts were thawed and diluted either four-fold for CS-12, CU, and TAM, or diluted two-fold for CS-24 with filtered water on the day of the test session, to make up to a total of 200 mL of “diluted aqueous extract”.The study meals (including rice) were prepared fresh every morning, and the test extracts were either added into rice (R) during cooking or made up into a drink (D). The study meals were rich in carbohydrates, providing approximately 50 g of available carbohydrates mainly from high-GI glutinous rice  and 20 g of cooked garden peas . The control meal (CON) was made up of 65 g of glutinous rice cooked in 200 mL of filtered water in a household rice cooker, 20 g of peas (steamed for 2 min in microwave), and a glass (200 mL) of filtered water. The rice (R) test meals consisted of 60 g of glutinous rice cooked in 200 mL of the diluted aqueous extract (detailed above), 20 g of peas, and 200 mL of filtered water, whereas the drink (D) test meals consisted of 60 g of glutinous rice cooked in 200 mL of plain filtered water, 20 g of peas, and 200 mL of diluted aqueous extract. The food ingredients used and the energy content and nutrient composition of each test meal as obtained from food packet labels or nutrient databases (US Department of Agriculture) are shown in g for 5 min at room temperature to obtain the supernatant. Subsequently, 100 µL of tap water (blank), supernatant, or gallic acid standard was added to 200 µL of 10% FC reagent in a 96-well clear polystyrene microplate followed by the addition of 800 µL of 700 mM sodium carbonate. After a 2-h incubation at room temperature in the dark, the absorbance at 765 nm was measured in a microplate reader . The TPC was calculated using the regression equation derived from a gallic acid standard curve and expressed as gallic acid equivalents (GAE).Total polyphenol contents (TPCs) were determined by the Folin–Ciocalteu (FC) assay in triplicates using a method adopted from Medina-Remón et al. . BrieflyThis was a randomized crossover design whereby each volunteer came for five or nine test sessions with a minimum 1-day wash-out between sessions. The order of the study meals were randomized using the RAND function in Microsoft Excel. Volunteers were asked to avoid strenuous exercise and alcohol intake 24 h prior to their test session and to arrive at the center at 08:30 after an overnight fast of at least 10 h. Upon arrival, volunteers had their baseline (T0) blood pressure measured and a fasted blood sample drawn (after 5 min of rest) at the antecubital vein via cannula by a trained phlebotomist. Volunteers were then served the study meal and instructed to consume the meal within 15 min. Subsequent blood collection was carried out at T15, 30, 45, 60, 90, 120, 150, and 180 min after the first bite. Duplicate blood pressure measurements were taken hourly at T60, 120, and 180 min. A schematic of the study design is shown in 2EDTA vacutainers , placed immediately on ice, and centrifuged at 1500× g for 15 min within an hour of blood draw to obtain plasma. Plasma samples were then stored at −80 °C in aliquots until ready for analyses. Plasma glucose and insulin concentrations were determined by the COBAS c311 and e411 automated analyzers , respectively. For each analyzer, a successful two-point calibration was performed at the start of the study. Thereafter, quality control was done daily before analyzing the plasma samples. For each assay, 250 µL of thawed undiluted plasma was analyzed in a Hitachi cup using the manufacturer’s recommended cassette for glucose and insulin measurement. All samples were analyzed over a 5-day period using the same analytical instrumentations and laboratory consumables . All samples from a given individual  were analyzed within the same batch and the same day, in order to limit any effects of experimental variations. The inter-batch coefficient of variation for glucose analysis was 7.51%, and that for insulin analysis was 1.99%. Venous blood was collected in KThe primary outcome of this study was the acute effects of the test ingredients on the postprandial glucose and insulin responses for up to 180 min. The secondary outcomes were: i) the effect of the form in which the test ingredients were administered (R vs. D) on the 30, 120, and 180 min postprandial glucose and insulin responses; and ii) the acute effects of the test ingredients on the postprandial blood pressure measured over 180 min. Incremental areas under the curve (iAUCs) of glucose and insulin were calculated using the change in glucose or insulin above baseline fasting concentration, ignoring the area beneath the baseline. Total areas under the curve (tAUCs) of systolic and diastolic blood pressure measurements were calculated using the absolute blood pressure values measured at the time points.All analyses in this study were done using Statistical Package for the Social Sciences (SPSS) version 24 . Data were assessed for normality using the Shapiro–Wilk test as well as visually from the histogram and normal Q–Q (quantile-quantile) plots. Square-root transformation of the data was used where necessary to achieve normality. The treatment effect on the iAUCs of insulin and glucose was tested using linear mixed effects procedure in SPSS with treatment as the fixed factor and compound symmetry (CS) covariance structure. For the interaction between form and treatment test, a linear mixed effects procedure with both treatment and form as fixed factors was used with CS covariance structure. Mean total polyphenol content (TPC) was lowest in the cumin aqueous extract (103.0 ± 3.4 mg GAE/100 g of cumin powder) and highest in the tamarind aqueous extract (440.1 ± 13.3 mg GAE/100 g of tamarind pulp). Mean TPC of corn silk was 233.1 ± 2.2 mg GAE/100 g of dried corn silk.Of the 18 volunteers enrolled in the study, 16 volunteers completed all nine test sessions. One volunteer only completed three test sessions, while another only completed five sessions. The baseline anthropometric and metabolic characteristics of the volunteers to indicate the profile of volunteers who completed the study are presented in 30, iAUC120, or iAUC180), as shown in 30, iAUC120, or iAUC180). However, there was a significant increase (p < 0.01) in postprandial insulinemia (iAUC120 or iAUC180) when the test ingredients were consumed as a drink (D) as compared with the same ingredients being cooked within rice (R), irrespective of the test ingredients. It should also be noted that the form × treatment interactions were not significant across all measures, as shown in The postprandial changes in systolic and diastolic blood pressures are shown in jeera” (cumin) rice), curries, soups  or as beverages such as “jal jeera” (cumin water in India) or as “nam makham” (tamarind drink in Thailand). Given their extensive consumption in several different forms , we further investigated any potential differences in their glycemic/insulinemic responses when either consumed as a drink or added into rice while cooking. Corn silk extract (CS) on the other hand was investigated not only due to its extensive use by traditional Chinese medicine practitioners as an anti-diabetic formulation [Given that Asians are at a greater risk of type 2 diabetes and prediabetes, and considering that the dietary carbohydrate loads in Asians are much higher than in other parts of the world, there is a drive to identify functional ingredients within the Asian dietary context which can improve glycemic control. This trial was set out to test the effects of three separate ingredients, at dietary doses, for their individual effects on postprandial glycemia and insulinemia when consumed along with a carbohydrate-rich meal consisting of a portion of high-GI rice. We have previously shown improvements in postprandial glycemic control with polyphenol-rich mixed spices ,26. The mulation ,40, but mulation .Despite our careful selection of the ingredients tested, our study failed to demonstrate any significant effects on postprandial glycemia or insulinemia as compared to the control meal without these ingredients. There could be several reasons for this observation. First of all, we recruited non-diabetic, healthy individuals, all of whom are likely to have had adequate glycemic control. Indeed, some studies have previously shown that the postprandial glycemic response even between high- and low-GI meals did not differ in young healthy individuals . FurtherWhile the phytochemical-rich ingredients used in our study per se did not give rise to any obvious effects on postprandial glycemia or insulinemia, there was a significant lowering of the postprandial insulinemic responses when the test meals were consumed within cooked rice (R) as compared to being consumed in the drink (D) form separately from the rice. This suggests that the food matrix in which phytochemical-rich ingredients are consumed has an effect on postprandial metabolic responses. Our research group has previously shown that both postprandial glycemic  and lipiSimilar to our findings, others have also reported greater postprandial insulinemic responses to a homogenized meal as compared with a solid meal . There cThis study demonstrated that adding aqueous extracts of corn silk, cumin, and tamarind at dietary doses, in two separate forms, to a high-GI rice consisting of approximately 50 g of available carbohydrates in healthy volunteers conferred no additional benefits on postprandial glycemia or insulinemia. Our randomized, controlled clinical trial findings are insightful and advantageous in that these ingredients are traditionally believed in Asia to be beneficial in reducing the risk of diabetes, although to date, the majority of the previous evidence supporting these claims had been undertaken in animals, most often at relatively high doses. Some of the limitations of this study were the acute nature of the study design, the limited number of postprandial parameters being measured, and the fact that this study was undertaken in a cohort of healthy, normoglycemic, non-diabetic individuals. While other longer-term beneficial effects  of the test ingredients in a population with a compromised glucose homeostasis  cannot be ruled out, this study highlights the need for more controlled clinical trials in the future before specific claims are assumed regarding the specific benefits of traditional ingredients. Finally, this study also highlights the importance of the food form on postprandial insulinemia, in that phytochemical-rich ingredients in liquid forms  may elicit greater postprandial insulinemic response than when consumed within a solid meal. Therefore, future applications of our study findings include the manipulation of food forms via various food consumption, preparation, and processing methods, in order to improve the metabolic consequences of certain foods and/or composited dishes.


Ph1 locus. Plant material was collected at early prophase, at the transition leptotene-zygotene, when the telomere bouquet is forming and synapsis between homologs is beginning. The six genotypes exhibit different levels of synapsis and chromatin structure at this stage; therefore, recombination and consequently segregation, are also different. Unexpectedly, our study reveals that neither synapsis, whole genome duplication nor the absence of the Ph1 locus are associated with major changes in gene expression levels during early meiotic prophase. Overall wheat transcription at this meiotic stage is therefore highly resilient to such alterations, even in the presence of major chromatin structural changes. Further studies in wheat and other polyploid species will be required to reveal whether these observations are specific to wheat meiosis.Polyploidization is a fundamental process in plant evolution. One of the biggest challenges faced by a new polyploid is meiosis, particularly discriminating between multiple related chromosomes so that only homologous chromosomes synapse and recombine to ensure regular chromosome segregation and balanced gametes. Despite its large genome size, high DNA repetitive content and similarity between homoeologous chromosomes, hexaploid wheat completes meiosis in a shorter period than diploid species with a much smaller genome. Therefore, during wheat meiosis, mechanisms additional to the classical model based on DNA sequence homology, must facilitate more efficient homologous recognition. One such mechanism could involve exploitation of differences in chromosome structure between homologs and homoeologs at the onset of meiosis. In turn, these chromatin changes, can be expected to be linked to transcriptional gene activity. In this study, we present an extensive analysis of a large RNA-seq data derived from six different genotypes: wheat, wheat–rye hybrids and newly synthesized octoploid triticale, both in the presence and absence of the  Polyploidization, or whole genome duplication (WGD), has an important role in evolution and speciation, particularly in plants. It is now clear that all seed plants and angiosperms have experienced multiple rounds of WGD during their evolutionary history and are now considered to possess a paleopolyploid ancestry . PolyploMeiosis is the specialized cell division that generates haploid gametes for sexual reproduction. During meiosis, homologous  chromosomes synapse along their length and recombine, leading to novel combinations of parental alleles, and ensuring proper chromosome segregation. Restriction of synapsis and crossover (CO) formation to homologous chromosomes (homologs) is therefore a prerequisite for regular meiosis. Subsequent recombination is also critical, not only to generate new combinations of genes, but also to ensure an equal distribution of genetic material and maintain fertility and genome stability across generations. One of the problems of polyploidization is that it is initially accompanied by irregular meiosis, due to the presence of more than two identical homologs in autopolyploids, or very similar chromosomes (homoeologs) in allopolyploids. Thus, one of the biggest challenges faced by a new polyploid, is how to manage the correct recognition, synapsis, recombination, and segregation of its multiple related chromosomes during meiosis, to produce balanced gametes.T. aestivum, , also known as bread wheat, is a relatively recent allopolyploid, with three related ancestral genomes, which although different, possess a very similar gene order and content. Hexaploid wheat has a 16 Gb genome size, with high similarity between homoeologous genomes in the coding sequences (95–99%), and with a large proportion of repetitive DNA (>85%)  . Despiter genome . Therefor genome . It woulPh1 (Pairing homoeologous 1), a dominant locus on chromosome 5B , they do synapse to the same level after the telomere bouquet has dispersed, whether or not Ph1 is present  within this cluster is responsible for both promotion of homologous CO and restriction of homoeologous CO, and is involved in improved synapsis efficiency , both in the presence and absence of Ph1. These six genotypes provided a unique set of transcription data, which can also be exploited in further studies. Surprisingly the analysis revealed that neither the level of synapsis, the ploidy level, nor the Ph1 locus affected overall meiotic transcription during the leptotene-zygotene transition stage.In this study we undertook a comprehensive study of transcription during early meiotic prophase, specifically at the leptotene-zygotene transition stage, when the telomere bouquet is formed in wheat and synapsis between homologs begins, to assess the effect on transcription of: changes in chromatin structure upon homologous recognition, level of synapsis, ploidy level, and presence of the Triticum aestivum cv. Chinese Spring , either containing or lacking the Ph1 locus ; wheat–rye hybrids crosses between hexaploid wheat either containing or lacking the Ph1 locus, and rye; octoploid triticale × Triticosecale Wittmack (2n = 8× = 56), obtained after genome duplication of wheat–rye hybrids either containing or lacking the Ph1 locus.The plant material used in this study and its production is described in Figure h1 locus ; rye SecT. aestivum cv. Chinese Spring or T. aestivum cv. Chinese Spring ph1b mutant  for the RNA-seq experiments or fixed in 100% ethanol/acetic acid 3:1 (v/v) for cytological analysis of meiocytes.Seeds were germinated on Petri dishes for 3 to 4 days. The seedlings were vernalized for 3 weeks at 7°C and then transferred to a controlled environment room until meiosis, under the following growth conditions: 16 h light/8 h night photoperiod at 20°C day and 15°C night, with 70% humidity. After 6 to 7 weeks, plants were ready for meiosis studies. Tillers were harvested after 6 to 7 weeks, at early booting stage, when the flag leaf ligule is just visible . Anthers were collected at early prophase, at the transition leptotene-zygote, which is during the telomere bouquet stage in wheat. For each dissected floret, one of the three synchronized anthers was squashed in 45% acetic acid in water to identify the meiotic specific stage. The two remaining anthers were harvested into RNAPh1 locus, and rye were collected as described in the “Plant Material” section. Three biological replicates were prepared for each genotype, so a total of 21 samples were obtained. Anthers at the selected meiotic stage were harvested into RNAlater . The anthers from three plants of each genotype were pooled in a 1.5-ml Eppendorf tube until 300 anthers were collected. As there were so few triticale seeds available, each triticale sample was derived from a single plant. Once sufficient anthers had been collected, the material was squashed using a pestle to release the meiocytes from the anthers, and the mix was transferred to a new Eppendorf trying to avoid as much of the anther debris as possible, to enrich the sample with meiocytes. The enriched meiocyte samples were centrifuged to eliminate the RNAlater and homogenized using QIAshredder spin columns . RNA extraction was performed using a miRNeasy Micro Kit  according to the manufacturer’s instructions. This protocol allows purification of a separate miRNA-enriched fraction  and the total RNA fraction (>200 nt) used in this study.Anthers from wheat, wheat–rye hybrids and triticale, both in the presence and absence of the TM Rapid Directional RNA-Seq Kit  with the NEXTflexTM DNA Barcodes–48  diluted to 6 μM. The library preparation involved an initial QC of the RNA using Qubit DNA  and RNA  assays, as well as a quality check using the PerkinElmer GX with the RNA assay . The constructed stranded RNA libraries were normalized and equimolar pooled into one final pool of 5.5 nM using elution buffer . The library pool was diluted to 2 nM with NaOH, and 5 μl was transferred into 995 μl HT1 (Illumina) to give a final concentration of 10 pM. Diluted library pool of 120 μl was then transferred into a 200-μl strip tube, spiked with 1% PhiX Control v3 and placed on ice before loading onto the Illumina cBot. The flow cell was clustered using HiSeq PE Cluster Kit v4, utilizing the Illumina PE_HiSeq_Cluster_Kit_V4_cBot_recipe_V9.0 method on the Illumina cBot. Following the clustering procedure, the flow cell was loaded onto the Illumina HiSeq 2500 instrument following the manufacturer’s instructions. The sequencing chemistry used was HiSeq SBS Kit v4 with HiSeq Control Software 2.2.58 and RTA 1.18.64. The library pool was run in a single lane for 125 cycles of each paired end read. Reads in bcl format were demultiplexed based on the 6 bp Illumina index by CASAVA 1.8, allowing for a one base-pair mismatch per library and converted to FASTQ format by bcl2fastq. RNA-seq data processing: the raw reads were processed using SortMeRNA v2.0 .One microgram of RNA was purified to extract mRNA with a poly-A pull down using biotin beads. A total of 21 libraries were constructed using the NEXTflexRNA v2.0  to remov2. The following formula was used to calculate the ratio (-1 to +1) of coverage; ratio = sample1–sample2/sample1+sample2.RNA-seq reads (trimmed non-rRNA reads as described above) for the 18 samples were aligned to the RefSeqv1.0 assembly , using HPh1 locus, by pseudoaligning the raw reads for these six samples against the Chinese Spring RefSeqv1.0+UTR transcriptome reference  were retained for differential expression analysis  were mapped to the in silico wheat–rye transcriptome using the same method as described above for wheat samples. Only genes with a mean expression >0.5 TPM in at least one condition  were retained for differential expression analysis. This included 83,202 genes in total, of which 50,292 were high confidence (HC) wheat genes, 22,138 were low confidence (LC) wheat genes and 10,772 were rye genes. Two comparisons were carried out to examine the effect of Ph1 , and one comparison performed to examine the effect of different synapsis levels and chromosome doubling . Differentially expressed genes for each comparison were identified using DESeq2 as described above, using three replicates per condition. The numbers of differentially expressed genes at various thresholds  were obtained for wheat and rye genes. GO term annotation was available for the wheat genes, therefore we focused only on differentially expressed wheat genes for GO enrichment analysis and excluded differentially expressed rye genes from this analysis. GO enrichment analysis was carried out as described for the wheat samples, again separately analyzing genes up-regulated >twofold with padj < 0.01, and genes down-regulated >twofold with padj < 0.01.Differential expression amongst wheat–rye hybrid and triticale samples was analyzed by constructing an eference  with theeference . In totaThe rye genome sequence is still incomplete; therefore, the present study focused on wheat-specific transcription effects. However, the rye data (PRJEB25586) are deposited to facilitate future analyses by the community.Ph1 used for the RNA-seq was sterile, so instead, another triticale lacking Ph1 which had 57 chromosomes, was used.It was not possible to analyze the samples used for the RNA-seq analysis, so their progeny were analyzed instead. One spike of every plant used for the transcription analysis was selfed, and three individuals from each progeny analyzed. One of the triticale lacking in situ hybridization (GISH) was carried out as described previously  by nick translation as described previously  according to the manufacturer’s instructions. Biotin-labeled probes were detected with Streptavidin-Cy5 . Digoxigenin-labeled probes were detected with anti-digoxigenin-fluorescein Fab fragments (Sigma).The preparation of mitotic metaphase spreads and subsequent genomic eviously . Meioticeviously . S. cereeviously . T. urarhttps://imagej.nih.gov/ij/) and Adobe Photoshop CS4  version 11.0 × 64.Images were acquired using a Leica DM5500B microscope equipped with a Hamamatsu ORCA-FLASH4.0 camera and controlled by Leica LAS X software v2.0. Images were processed using Fiji  under project number PRJEB25586. Analyzed data for the wheat samples (TPM and counts) were integrated in the expVIP platform www.wheat-expression.com . Although our aim in these samples was principally to study the expression of wheat genes, the use of a hybrid transcriptome reduced the possibility of mis-mapping between rye and wheat reads, which would have led to inaccurate quantification of expression. Kallisto was used for mapping because in wheat, it accurately distinguishes reads from homoeologs carrying genes with a sequence identity between 95–97%  and triticale an octoploid , and although vegetative development is normal in both genotypes, the haploid hybrids are completely sterile. The reason for sterility in the wheat–rye hybrid is that meiosis is highly compromised, with only one CO at metaphase I and subsequent random segregation of chromosomes. Despite this, wheat transcription during early meiotic prophase seems not to be affected.At the leptotene-zygotene transition, when the telomere bouquet is tightly formed, only homologous chromosomes can synapse. In wheat–rye hybrids there are no homologs present, and therefore, no synapsis takes place at this stage; whereas in triticale, a significant level of synapsis occurs. However, no overall change in wheat transcription was observed when these two genotypes were compared Figure . Even moPh1 locus was made : a terminal region in 1DL and 3DL, and a large distal region on 5BL. Interestingly, every one of these chromosome regions corresponded to a chromosome deletion on a homoeologous chromosome. For example, the terminal deletion on 1AL, corresponded to the terminal increased transcription on 1DL. Recombination could occur between 1A and 1D in the absence of Ph1, resulting in two 1D chromosomes plus two 1A chromosomes being detected in this material, in which the distal part of 1AL had a chromosome segment from 1DL. This, therefore, resulted in four copies of the D-genome chromosome segment, and hence increased transcription. These observations are consistent with wheat nulli-tetrasomic line analysis  were expressed in our samples, of which 72,430 were from wheat and 10,772 from rye . In the case of genes up-regulated upon chromosome doubling , two thirds of the GO terms were related to stress and response to external stimuli, the rest being related to cell communication and catabolic processes. Although the present study focuses on changes in overall gene expression, we also extracted the functional annotation for all DEG, available at Supplementary Table To check whether DEGs genes were associated with common processes, Gene Ontology (GO) term enrichment was carried out on genes differentially expressed between samples , 65,583 were expressed in our wheat samples, from which 474 genes (0.72%) were differentially expressed when Ph1 was deleted  genes were differentially expressed for triticale lacking Ph1  is a member of the SWI2/SNF2 family known to be involved in DNA repair and recombination in somatic tissue, while no function during meiosis has been reported  was obtained and estimated to be of 70 Mb in size. Using our gene expression data and the RefSeqv1.0 assembly, the ph1b deletion is now defined to a 59.3 Mb region containing 1187 genes, from which 299 genes are expressed in our RNA-seq data. The locus was further defined to a smaller region (ZIP4 gene (TaZIP4-B2), which has been recently identified as the gene responsible for both promoting homologous CO and restricting homoeologous CO  and one on 5D, claiming that only the 5B copy was metaphase I-specific and therefore, responsible for the phenotype characteristic of Ph1. However, blasting these gene sequences against the RefSeqv1.0 assembly showed that 5Balt was a fourth gene copy located on 5A chromosome just upstream of the original 5A copy (5A-1: TraesCS5A01G381600LC and 5A-2: TraesCS5A01G381700LC). The RNA-seq data obtained in the present study, as well as 849 wheat RNA-seq samples now publicly available  is the dominant ZIP4 copy and it is expressed in all tissue types, including all meiotic stages. C-Ph1 has an almost tissue-specific expression pattern, limited to stamen tissue during the heading stage (post-meiosis stage), and with the 5D copy being expressed dominantly over all other gene copies . None of the C-Ph1 copies is expressed during any meiosis stage, except for the 5D copy that is expressed at a very low level (<4 TPM) in comparison with its expression in anthers at heading (>1700 TPM). In summary, we can confirm that C-Ph1 on 5B is not expressed during meiosis, and cannot therefore be responsible for any Ph1 effect during metaphase I.In 1977 , the ph1r region , now defogous CO , 2018a. aphase I ; howevervailable  and 8 whPh1 locus, has been well documented in previous studies , with a chromosome number of 56, and possessing 14 chromosomes from the A, B, and D genome, plus 14 chromosomes from rye  has recently been released by the International Wheat Genome Sequence Consortium , giving Ph1. All plant material was collected during early prophase, at the leptotene-zygotene transition, coinciding with telomere bouquet formation and synapsis between homologs. We addressed three questions in the study: whether overall wheat transcription was affected by the level of synapsis (and chromatin structure changes at the time of homolog recognition); whether wheat transcription was reshaped upon genome duplication; and whether wheat transcription was altered in the absence of the Ph1 locus. Surprisingly, the answer to all three questions was negative. Wheat transcription was not affected in any of the three situations, revealing an unexpected level of transcription stability at this very important developmental stage. These results contrast with observations in somatic tissue of resynthesized hexaploid wheat, where 16% of genes were estimated to display non-additive expression  chromosomes pair and then synapse through the polymerization of a protein structure known as the synaptonemal complex, which provides the structural framework for recombination to take place. Throughout the whole of meiosis, but particularly during the synaptic process, there are multiple changes in chromatin structure and organization (and even positioning in the nucleus), taking place within a relatively short period of time and needing to be highly regulated. In wheat, synapsis is initiated during the telomere bouquet stage at early prophase, during the transition leptotene-zygotene . MoreoveSurprisingly, however, we did not find the expected differences in overall wheat transcription and gene expression when comparing samples with different levels of synapsis, indicating that the structural changes associated with this process were not directly coupled to transcription. Probably the clearest example of this was the comparison of wheat–rye hybrids and octoploid triticale. Wheat–rye hybrids possess a haploid set of wheat and rye chromosomes (there are no homologs present) and no synapsis is observed during the telomere bouquet stage . In contEven more striking is the fact that in our study, global gene expression was not affected by WGD. Several previous studies have reported genetic and epigenomic processes being disrupted after hybridization and polyploidization, with subsequent changes in gene expression . These sWe propose that a possible explanation for the striking robustness in gene expression during early meiotic prophase is that the transcription of genes required for the meiotic program has already occurred prior to the leptotene-zygotene transition. Meiosis is a very complex process which takes place in a relatively short period of time. In wheat, the whole meiosis process lasts only 24 h at 20°C, with the whole process of synapsis being less than 6 h long . TherefoPh1 locus in wheat is by far, the best characterized locus involved in the diploid-like behavior of polyploids during meiosis. Ph1 has a dual effect during meiosis: firstly, improving the efficiency of homologous synapsis and secondly, preventing CO formation between homoeologs while increasing CO between homologs (ZIP4 gene inside the Ph1 locus on 5B (TaZIP4-B2) has been identified as responsible for the effect of this locus on recombination and suggested to be also involved in the improved synapsis efficiency  and a fourth copy on chromosome 5B (TaZIP4-B2). TaZIP4-B2 is a transduplication of a chromosome 3B locus (Ph1 locus upon polyploidization. In wheat lacking Ph1 (and therefore TaZIP4-B2), ZIP4 copies on the homoeologous group 3 are still present, allowing CO formation, even if a small fraction occurs between non-homologous chromosomes. In the case of an allopolyploid species such as wheat, the process of homolog recognition is further complicated compared to diploids, by the presence of homoeologous chromosomes. We can hypothesize that upon polyploidization, the newly formed hexaploid wheat already had mechanisms in place for the meiotic sorting of homologs from homoeologs during the telomere bouquet. This would have provided the new allopolyploid with some fertility until improved by the transduplication of ZIP4 from 3B into the Ph1 locus, with further modification and stabilization of the meiotic process. Over time and evolution, the efficiency and stability of meiosis could be completely established. The transduplication of ZIP4 is an example of how the meiotic program could be modified in polyploids in general, and wheat in particular, during evolution. It also illustrates the requirement to study such processes directly in these crop species, where there is a potential for manipulation in breeding programs.The homologs . Recentlficiency , 2018a. synapsis . Althougsynapsis . In dipl3B locus  and mostC-Ph1, which is a syntenic ortholog of the RA8 gene in rice, has also been proposed to contribute to the Ph1 effect on recombination  is by far the most dominantly expressed. In addition, the newly generated wheat genome assembly reveals that the VIGS hairpin construct used for C-Ph1 silencing (C-Ph1 copy on 5B shows no meiotic expression (<0.5 TPM), being mostly expressed afterward during pollen formation. The C-Ph1 copy on 5D is expressed during meiosis at a very low level (<4 TPM) compared to its expression in anthers during the heading stage (>1700 TPM), exhibiting a similar expression profile to the C-Ph1 ortholog in rice RA8. These observations explain why our deletion covering the 5B copy of C-Ph1 did not exhibit meiotic phenotype or sterility , being the same level of homoeologous CO to that observed in ph1b deletion mutants , of which 186 were located within the region corresponding to the 5B deletion, and 139 were located within the regions corresponding to the 3A and 3B deletions present in all Ph1 samples. Therefore, no major global changes in wheat expression were observed in wheat lacking Ph1 during early prophase. The effect of the CDK2-like genes inside the Ph1 locus on premeiotic replication and the associated effects on chromatin and histone H1 phosphorylation did not subsequently affect overall gene expression in early meiotic prophase. Moreover, the significant structural changes observed in the absence of Ph1  were not associated with changes in global gene expression. This result is consistent with our previous conclusion that gene expression during meiotic early prophase is very stable and quite resilient to changes in chromatin structure.In the present study we used a total number of 21 different Ph1 (or TaZIP4-B2) was only 4–5 COs fewer than in wild type wheat . These lines do not currently exhibit rearrangements, but will probably also accumulate reorganizations in further generations, so we recommend checking and cleaning the lines periodically.The karyotypic instability in the absence of reported , but the in 1977  and sincPh1) is unstable meiotically, with variable frequency of univalents in metaphase I and reduced fertility (Ph1 locus in the wheat genome plays the same role in this new species, ensuring only homologous recombination. It has previously been speculated that late DNA replication of rye heterochromatin interferes with chromosome synapsis when rye chromosomes are placed in a wheat genetic background (Octoploid triticale is the synthetic amphiploid resulting from the chromosome doubling of the hybrid between hexaploid wheat and rye. It is therefore, a new allopolyploid species. Primary octoploid triticale (containing ertility . For ourckground ; howeverckground . There iPh1, the locus responsible for the diploid-like behavior of hexaploid wheat. As in the case of triticale containing Ph1, only plants with 56 chromosomes were selected for the RNA-seq analysis. The fertility of these triticale plants lacking Ph1 was extremely low, ranging from nine seeds to complete sterility. GISH analysis on both somatic and meiotic cells showed that unlike in triticale containing Ph1, there were extensive reorganizations resulting from non-homologous recombination in the triticale lacking Ph1. Even though these triticale plants had only gone through three meiotic events since their synthesis, there were extensive recombination events between homoeologs. Chromosome fragmentation was also detected, particularly involving rye. There is a difference in heterochromatin DNA replication in the absence of the Ph1 locus in wheat–rye hybrids (Ph1, the additional delayed replication produced by the absence of Ph1 could affect excessive DSB formation, causing not only a decrease in CO formation but also lack of DSB repair, and therefore, chromosome fragmentation. In any case, triticale lacking Ph1 exhibits even more chromosomal rearrangements than wheat lacking Ph1, leading to an extreme phenotype and sterility.We also assessed the consequence on meiosis of generating triticale in the absence of  hybrids . If, as Ph1 locus. This suggests that the transcription of genes required for early meiotic prophase has already occurred prior to this stage. Genetic studies in polyploids such as wheat have lagged far behind diploid species, partly because of the lack of key genetic resources. However, the release of the RefSeqv1.0 assembly in hexaploid wheat (www.wheat-expression.com (Understanding polyploidization is of great importance in the understanding of crop domestication, speciation, and plant evolution. One of the biggest challenges faced by a new polyploid is how to manage the correct recognition, synapsis and recombination of its multiple related chromosomes during meiosis, to produce balanced gametes. In the last few years, there has been a better understanding of the meiotic process from studies of diploid plants and other model organisms . Polyploid wheat , the avasion.com  enablingsion.com , will noAM, PS, and GM conceived and designed the study. AM obtained the hybrids and triticale, produced the meiotic RNA samples, did the cytological analyses, interpreted the results, and wrote the first draft of the manuscript. PB carried out the differential expression analysis, assisted with analysis of all data, and contributed corrections and suggestions. JH did the mapping using HISAT and created the chromosome coverage plots. AA carried out some data analysis and assisted with analysis of data. RR-G carried out the mapping using kallisto for the differential expression analysis and integrated the wheat analysis into the expVIP platform. PS and GM interpreted the data. CU contributed in corrections and suggestions. GM edited the manuscript. All authors have read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Saccharomyces, Candida, and Cryptococcus yeasts (and their hybrid offspring) and analyse associations with chromosome size and specific stressors. We discuss how aneuploidy, a segregation error, may in fact provide a natural route for the diversification of microbes and enable important evolutionary innovations given the right ecological circumstances, such as the colonisation of new environments or the transition from commensal to pathogenic lifestyle. We also draw attention to a largely unstudied cross link between hybridisation and aneuploidy. Hybrid meiosis, involving two divergent genomes, can lead to drastically increased rates of aneuploidy in the offspring due to antirecombination and chromosomal missegregation. Because hybridisation and aneuploidy have both been shown to increase with environmental stress, we believe it important and timely to start exploring the evolutionary significance of their co‐occurrence.Aneuploidy is the loss or gain of chromosomes within a genome. It is often detrimental and has been associated with cell death and genetic disorders. However, aneuploidy can also be beneficial and provide a quick solution through changes in gene dosage when cells face environmental stress. Here, we review the prevalence of aneuploidy in  In resp2.3.1S. cerevisiae is generally not harmful to humans, it can become an opportunistic pathogen in immunocompromised individuals. A study of 136 such strains found that 36% contained aneuploidies , octoploid (8N), or even higher ploidy. Under stressful conditions, these titan cells can produce near diploid daughter cells, with aneuploidies affecting different chromosomes. These aneuploid cells often show resistance to different drugs. Chr I aneuploidy was found to occur more often (in 26 out of 53 strains), but other chromosomes also assisted with fluconazole resistance  and Chagas disease (Trypanosomacruzi). Aneuploidy is particularly widespread across Leishmania and has been shown to vary from few aneuploid chromosomes , with tetraploid strains containing significantly higher rates of aneuploidy than lower ploidy strains  the type of stress, (b) the degree of stress, and (c) the rate at which it is applied. For example, aneuploidy in 4S. cerevisiae × S. paradoxus, which differ at about 14% of all nucleotides in their genomes  frequently found in the wild  in 40 different environments found extensive transgressive segregation , suggesting that hybridisation may play a significant role in the phenotypic evolution of this important human pathogen  showed signs of aneuploidy . This includes aneuploidies reported by each reference, as well as the total aneuploidies, ordered by size of chromosome.Click here for additional data file.Table S2Tables containing the aneuploidies used for analysis of chromosomes size and rate of aneuploidy separated into four categories. A) Industrial strains , B) pathogenic strains , C) wild strains  and experimental strains . This includes aneuploidies reported by each reference, as well as the total aneuploidies, ordered by chromosome size. Industrial strains include those linked with beer, wine, other fermentation and bioethanol production. Pathogenic strains are those found in clinical and other human samples. Wild strains are those found on plants, in insects, in water and other natural settings. Experimental strains are classified as strains involved in laboratory experiments, in which strains start as clonal and are studied over time. This includes a hybridisation experiment, studies of a deletion collection and studies of adaptation to starvation. 18 strains were uncategorised and are not included in these tables. These uncategorised strains are included in Supplementary Table 1 and Figure 2.Click here for additional data file.Table S3.Table containing the data used for analysis of ploidy and proportion of aneuploid strains . Proportions are separated by individual references and the mean of all proportions for each ploidy.Click here for additional data file.Figure S1.Scatter plots of the data from Supplementary Tables 2. This includes industrial strains (A), pathogenic strains (B), wild strains (C) and experimental strains (D). Regression line indicates significant correlation. Analyses and graphs were made using R version 3.5.1 (Feather Spray; R Core Team, Click here for additional data file.


Telocytes (TCs) are a distinct stromal cell type described in many organs. The present study investigated the existence of TCs within the efferent ductules in camel and the changes that occur in their morphology and activity during active and inactive reproductive seasons. TCs in the camel had a cell body and multiple telopodes (TPs), and most TCs had indented nuclei that exhibited prominent intranucleolar chromatin. TCs exhibited seasonal differences which were evaluated by histochemistry, immunohistochemistry (IHC), Transimition electron microscopy (TEM) and scanning electron microscopy (SEM). The presence of TCs in camel efferent ductules has been confirmed by CD34 positive immunostaing. In addition to the expression of the vascular endothelial growth factor (VEGF) which was stronger in the summer season. TCs exhibited stronger immunoreactivity for progesterone and oestrogen alpha receptors (ESR1) in the spring than in the summer. In addition, TCs showed strong positive immunostaining for both vimentin and androgen receptor (AR). Several ultrastructural changes were observed in TCs during the two seasons. TPs in the summer season had delicate ramifications whereas, in the spring, TPs displayed fine arborization and became more corrugated. TCs acquired signs of exaggerated secretory activities in the spring; TPs became expanded and packed with secretory vesicles. Thus, we conclude that, hormonal alterations during the reproductive cycle impact the morphology and secretory behavior of TCs. They establish a cellular communication system and play a central role in the functional regulation of different types of cells and structures5. TCs have unique morphological features that distinguish them from other stromal cells. TCs possess cell prolongations; telopodes (TPs) which extend from the cell body. TPs form an interstitial labyrinthine network to allow cellular communication. TPs have thin segments or podomeres and interval expansions or podoms which are rich in mitochondria, endoplasmic reticulum, and caveolae6.Telocytes (TCs) represent a distinct type10 and are involved in mechanoreception11. TCs also have excitatory and inhibitory neurotransmitter receptors12. The role of these cells in organ regeneration has been studied in several organs, including the heart, lung, skeletal muscle, skin, meninges and choroid plexus, eye, liver, uterus, and urinary system13. Based on gene expression analyses, various functions have been suggested for TCs such as cellular signalling15, cell expansion and movement14, tissue homoeostasis, remodelling15 and repair13, embryogenesis16, morphogenesis17, angiogenesis15, suppression of oxidative stress and cellular ageing18, and protection against inflammation and oncogenesis19.Several broad communication functions have been described for TCs. TCs transmit nerve impulses to smooth muscle cells20. The types of cell contacts in TCs including direct apposition of the cell membranes of adjacent TCs, adherence , and gap junction. Gap junctions play a significant role in signal transduction between cells21. The paracrine functions of TCs depend on molecular transfer through extracellular vesicles; exosomes, ectosomes and multivesicular vesicles23.Cellular connections are prominent feature of TCs that provide functional support to other cells. Two modes of communication are described for TCs; cell contact or through paracrine signaling. Cell contact is classified according to cell type into homocellular or heterocellular contact. TCs exhibit various forms of heterocellular contacts, including minute junctions such as point contacts, nanocontacts, planar contacts and cell contacts with an intermembrane distance, allowing macromolecules to interact24. In the present study, we investigated TCs changes in response to hormonal changes in the genital organs in one of the seasonal breeder animals (camel) during the active and inactive reproductive seasons. We used samples of the efferent ductules for the one-humped camel obtained in two different seasons, summer and spring. Efferent ductules are important for sperm transportation and fluid reabsorption25. Camel breeding activity reaches maximum levels during the active or rutting period; in the winter and spring seasons while the breeding activity declines during the inactive or non-rutting period; summer and autumn27. The main goal of the present investigation was to recognize TCs in the efferent ductules, their distribution, identify camel TCs characteristics, their relations to other cells, and explore the activity of TCs during inactive and active seasons of the reproductive cycle.Microenvironment- dependent changes in TCs behavior have been previously described; Hormonal administration affects the morphology and activities of TCsThe present study was performed on efferent ductules collected from 10 clinically healthy mature camels (Camelus dromedarius) during the spring season (March-April) and during the summer season, which represents the inactive period (July-August). The materials were collected from the Bani-Adie slaughter house in Assiut-Egypt.Efferent ductules were dissected and were fixed using (a) Bouin’s solution for light-microscopic examination and (b) Karnovsky fixative  for semithin sections and electron microscopy.Samples fixed in Bouin’s solution were extensively washed in 70% ethanol (3 times for 24 h) to remove the fixative prior to subsequent tissue processing steps for paraffin block preparation; Fixed samples were dehydrated in ascending grades of alcohols at 70%, 80%, 90% and 100% for 90 min at each concentration. The samples were cleared using methyl benzoate. Dehydrated samples were then impregnated and embedded in Paraplast . Serial sections of 3–5 μm were cut using a Richert Leica RM 2125 Microtome, Germany and mounted on glass slides. Sections were kept in an incubator at 40 °C.et al.28 with modification. Stock solution: 50 mg acridine orange is dissolved in 10 ml of distilled water and stored in the refrigerator, . The pH of the staining solution was approximately 3 and the AO concentration was 0.01%.The procedure was according to that of Hoff, The staining procedure: 5-μm paraffin sections were dewaxed (2 times for 30 min) and rehydrated in a descending series of ethanol  and DW. Dried sections of glass slides were fixed with methanol and dried in a trough with AO staining solution (0.01 per cent). After 2 minutes of staining, the slides are washed gently with DW, dried and then analyzed using a Leitz DM 2500 microscope with the external fluorescent unit Leica EL 6000.29 at 4 °C overnight. Semithin sections (1 μm) were cut using an ultramicrotome Ultracut E  and stained with toluidine blue30.Specimens from efferent ductules were used for semithin sections. Small pieces 2.0–3.0 mm in length were fixed in Karnovsky fixative31 according to the following protocol: paraffin-embedded tissue sections (5 μm) were dewaxed, rehydrated, and rinsed 3 times in PBS (pH 7.4) for 5 min. Endogenous peroxidase was inhibited by soaking the sections in 1% H2O2 for 10 min at room temperature, followed by intense washing under running tap water for an additional 10 min. For antigen retrieval, the slides were heated in water bath (for 20 min) in citrate buffer (pH 6.0) using a water bath followed by cooling for 20 min  was carried out in 0.1 M sodium citrate buffer solution (pH = 6) for 4 min using a microwave (600 Watt). The sections were incubated at room temperature for 2 h with blocking solution , 1% bovine serum albumin (Roth) and 0.3% Triton X-100 (Fluka Bio Chemika) to minimize non-specific labelling and increase the permeability to the efferent ductule tissue. Sections were incubated 24 h at 4 °C with primary antibody against vimentin  and androgen receptor marker . To visualize the primary antibody the sections were first washed with PBS (3 × 10 min each), followed by incubation 2hrs (in dark) with the same blocking solution containing the secondary antibody conjugated to Alexa-594 . Following a 1x PBS wash, sections were incubated for 10 min with DAPI  to visualize the nuclei, and FITC-coupled Tomatolectin  to visualize the lectins (carbohydrate-binding proteins). The sections were washed again with PBS (3 × 10 min each). Finally, the sections were cover slipped with fluoro mount mounting medium , and were kept in the dark to preserve fluorescence until examination with an AxioImager M2 fluorescent microscope (Zeiss) and pictures captured with AxioCam HRc camera.Representative specimens from the efferent ductules were washed several times in normal saline and then fixed in a mixture of 2.5% paraformaldehyde and 5% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.3, at 4 °C for 24 h. Thereafter the specimens were washed 4 times for 5 min in the fixation buffer and post fixed in 1% osmic acid in 0.1 M sodium phosphate buffer for additional 2 h at room temperature, followed by washing 4 times with 0.1 M sodium phosphate buffer for 15 min. The samples were dehydrated using different concentrations of alcohol; 50, 70, and 90% for 30 min at each concentration and 100% for 2 days  followed by isoamyl acetate for 2 days. The dehydrated samples were subjected to critical-point drying with a Polaron apparatus. Finally, the samples were coated with gold using JEOL- 1100 E ion sputtering device and observed with a JEOL scanning electron microscope (JSM 5400 LV) at 10 KV.Ultrathin sections were obtained by a Reichert ultra-microtome. The sections (70 nm) were stained with uranyl acetate and lead citrate and examined by a JEOL100CX II transmission electron microscope in the electron microscopy unit of Assiut UniversityScanned and transmission images were colored using the Photo Filter 6.3.2 program. To increase the visual contrast between several structures on the same electron micrograph.32. This has been done by the following steps: open image with CMEIAS Color Segmentation, then select “Process” from the menu items, and subsequently choose “Negative image34”.Negative images were performed using CMEIAS Color Segmentation, an improved computing technology used to process color images by segmenting foreground object of interest from the backgroundThe present study aimed to identify TCs in the camel efferent ductules and investigate the morphological changes of TCs and their secretory behavior in response to seasonal variation.TCs were identified in camel efferent ductules and, these cells were composed of the a cell body and TPs. TCs in camel efferent ductules were organized under the epithelium Figs  and 2A, The typical morphological features of TCs were identified for the first time using “Acridine Orange staining technique”. As observed in Fig. 1). Notably, ESR1 positive network was more complex in the spring than in the summer  that influence stem cells by paracrine or juxtracrine mechanisms56. Moreover, TCs established contact with blood vessels, glomus, nerve fibers and the epithelium. Hence, TCs likely serve as transducing centers that provide cell singling via a TPs network to other cell types and structures6.The communication between TCs and stem cells indicated a potential role for TCs in tissue regeneration. A similar conclusion was reported in the heart; after joining with stem cells, TCs are involved in the regeneration and repair of myocardial infarction57. These results may be attributable to hormonal variations between the two seasons. Gonadal activity is increased during the spring and declines during the summer in the camel27. Thus, TCs play an important role in male fertility. The impairment of oviduct TCs leads to infertility58.The present study described for the first-time changes in TCs behavior during the active and inactive breeding seasons in the efferent ductules of the camel. TCs exhibited moderate seasonal differences in their morphological features and activities. Some characteristics (morphology and density) of TCs change with some conditions42 as well as avian species59, reptiles50, amphibians60 and clitellates species2. CD34/PDGFR-α has been identified as a specific marker for TCs61. CD34 is a transmembrane phosphoglycoprotein that identified on hematopoietic stem and other progenitor cells including muscle satellite cells, corneal keratocytes, interstitial cells, epithelial progenitors, and vascular endothelial progenitors62. Vimentin positive TCs are also recognized in various tissues and organs such as uterine tube and uterus63, lung18, placenta64, mammary gland65, and heart66. Vimentin is one of the intermediate filaments provide structural support to maintain cellular integrity and protective function against stress67. Expression of vimentin is associated with normal development, cellular transformation, and growth in tissue culture68. Vimentin is expressed in a wide range of cells; fibroblasts, endothelial cells in blood vessels, epithelial cells, macrophages, cells of cartilage and bone, some vascular smooth muscle cells and melanocytes68. In addition, TCs exhibited stronger immunoreactivity for ESR1, PR and AR in the spring than in the summer. Steroids, particularly estrogen, progesterone and androgen mediated their actions through ESR, PR and AR70. Oestrogen plays a vital role in efferent ductule functions to reabsorb testicular fluid; thus, the lack of ESR1 may lead to a disturbance in the epithelial morphology and inhibit reabsorption of the testicular fluid71. However, progesterone affects male fertility and testosterone biosynthesis72. There is considerable evidence suggesting that, TCs are important targets for steroid hormones. TCs can function as ‘hormonal sensors’ in the human reproductive tract because they express progesterone and estrogen receptors8. Previous studies have reported the presence of ESR and PR on TCs of the myometrium73, fallopian tubes41, Seminal vesicles40. TCs in the fallopian and myometrium tissues may act as steroid sensors74. TCs may be involved in androgens secretion as reported before, they are connected to Leydig cells50.TCs share immunological marker characteristics of undifferentiated stem cells, such as CD34 and vimentin. In the current study, all TCs in the efferent ductules of camel were CD34, VEGF and vimentin positive. Camel TCs express CD34 similar immunophenotype of TCs in other mammalian species75, maintain vascular integrity76 and regulate vascular permeability77. Hence VEGF is termed as the vascular permeability factor (VPF). VEGF increase the vascular permeability by the endothelial cells through increasing the fenestrations78. Both VEGF and PDGF Receptor-α are expressed by TCs80. In the current study, VEGF was also expressed by camel efferent ductule TCs. The seasonal variation of TCs VEGF immunoreactivity was observed. TCs in the inactive summer season express strong immunoreactivity for VEGF more than the active spring season. Diminishing expression of the VEGF in the spring may serve in the thermoregulation that required for spermatogenesis. VEGFA have a significant role in the regulation of vascular permeability as well as in spermiogenesis and the proliferation of spermatogonia82.TCs could play a potential role in the development of blood vessels and endothelium. Vascular endothelial growth factor (VEGF) is one of the signaling protein belongs to the platelet-derived growth factor (PDGF) family. VEGF promote angiogenesis83 demonstrating that VEGF expression in the testis is downregulated during the mating season in one of the seasonal breeder animals, roe deer. Moreover, Camels have adapted to the hypoxic condition of the desert during summer season, in turn the hypoxia activates hypoxia inducible factor 1 (HIF-1). Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that involved in regulation oxygen consumption84. VEGF is one of the downstream gene of the HIF85. This may explain higher expression of the VEGF of TCs in the summer than the spring season.This result agree with earlier report86. TCs acquired signs of exaggerated secretory activities in the spring. TPs became expanded and packed by secretory vesicles. As observed by SEM, a large number of secretory masses were shed from TCs. Some TCs became enlarged in size. It suggests that during the spring season, elevation of the reproductive activity, there was increased demand for extensive transmission of intercellular information that utilizes small molecules, exosomes87. These vesicles may contain various growth factors or cytokines, which are very important in regulating the microenvironmental factors87.Several ultrastructural changes were observed in TCs during the both seasons. TPs in summer had delicate ramifications whereas, TPs in spring may exhibit fine arborization and became more corrugated. TCs, to become able to connect with the other cells or structures through their TPs or by extracellular vesicles and play a potential role in the maintenance of the reproductive tissue homeostasis and renewal88.Intranucleolar chromatin was observed in TCs as well as lymphocytes. The structural organization of intranucleolar chromatin is associated with seasonal and hormonal variation. This finding suggests that the intranucleolar chromatin may act as a temporarily inactive gene that is activated when necessary to satisfy the functional demand in the reproductive cycle particularly the secretory function. Identification of the intranucleolar chromatin composition revealed that they represented as rDNA transcriptional units89.In conclusion, seasonal differences in TCs morphology and behavior are likely regard to hormonal variations between the two seasons. Hormonal alternations during the reproductive cycle may influence the TCs morphology and secretory behavior. The results of the present study support the hypothesis that TCs are affected by microenvironmental changes. TCs gain additional short TPs in an environment characterized by oxidative stress. These cells also change their morphology and acquire a long and slender shape in N-acetyl cysteine cell culture mediumsupplementary figures and legends