Vibrio cholerae, the causative agent for cholera. The vaccine packets containing buffer powder and lyophilized CVD 103-HgR are reconstituted in water and consumed. Studies were performed to explore the viability of CVD 103-HgR in drinking waters from common sources. CVD 103-HgR vaccine was reconstituted in bottled and tap waters from the United States and Europe, and viability was measured via colony forming units assay. Chemical analysis of select water samples was used to identify chemicals that have a negative effect on CVD 103-HgR viability. CVD 103-HgR titers were stable in all bottled waters tested, including purified bottled water, bottled spring water, and sparkling waters. However, tap water from certain cities in the US and Europe affected viability and are not compatible with vaccine. Water chemistry revealed that these tap waters contained copper, likely leached from copper plumbing. These studies give high confidence in the stability of CVD 103-HgR reconstituted in a variety of bottled waters. Waters containing copper, including tap water, should not be used to reconstitute CVD 103-HgR strain oral vaccine due to the common use of copper plumbing.CVD 103-HgR live, attenuated oral cholera vaccine strain is indicated for single dose immunization against  Vibrio cholerae bacterium that is characterized by severe watery diarrhea, and vomiting that can lead to dehydration, shock, and death if left untreated1. There are several oral cholera vaccines available worldwide, the majority of which are whole-cell killed bacteria that require more than one dose2. PXVX0200 is a single dose live attenuated oral cholera vaccine containing the CVD 103-HgR vaccine strain of V. cholerae O1 Inaba 569B that is licensed under the tradename Vaxchora by the US Food and Drug Administration (FDA) and European Medicines Agency (EMA). Per the published instructions for use for Vaxchora vaccine4, the PXVX0200 vaccine is prepared by reconstituting the buffer and CVD 103-HgR active components in 100 mL of water. One dose of the vaccine consists of 4.5 g of buffer component  and 2 g of the active component  with a commercial specification of 4 × 108 to 2 × 109 colony forming units (CFU) per dose.Cholera is an enteric disease caused by the Gram-negative 5, but they can also show greater susceptibility to factors such as temperature and components in the vaccine mixture6, including the water used for reconstitution. Here, we describe a study exploring the compatibility of the cholera CVD 103-HgR strain with drinking water from different sources in the United States (US) and Europe in order to better characterize the vaccine and to ensure that the full potency is administered to the patient. Compatibility in this context is defined as maintaining viability of CVD 103-HgR, as measured by CFU/mL, within approved vaccine specification.Live vaccine strains, such as CVD 103-HgR have the advantage of producing a long-lasting immune response with a single dose by mimicking natural infection7. Drinking water in the United States (US) and European Union (EU) can be broadly divided into two categories: municipal (tap) water and bottled water. Municipal water is regulated by the Environmental Protection Agency (EPA) in the US9 and by the European Commission in the EU10. Bottled water is a specified type of drinking water that must meet additional standards and is regulated by the FDA in the US11 and by the European Commission in the EU12.PXVX0200 has the primary role of preventing travelers from developed nations from contracting and spreading cholera to vulnerable populations. In developed nations, drinking water is regulated by government agencies based primarily on guidelines published by the World Health Organization that describe permissible limits of chemical and biological contaminantsThis report provides data from studies performed on the compatibility of CVD 103-HgR live, attenuation cholera vaccine strain with drinking water from various sources, including tap water and bottled water from the US and Europe.13. Ascorbic acid, which is a major ingredient in the PXVX0200 buffer component, has been shown to effectively neutralize both chlorine and chloramine14. Pipes that carry the drinking water from the treatment plant to the point-of-use can also leach chemicals that are present when the water is collected from the tap15. The content of chemical compounds in tap water varies between municipalities that can be derived from the pre-treated water source, disinfecting and treatment methods, and drinking water infrastructure including household plumbing.The majority of public tap water is derived from surface and ground water, such as lakes, rivers, reservoirs, and underground aquifers. The water is processed through municipal water treatment plants by different chemical and physical methods depending on the source and quality of the water. Treatment plants use a combination of methods to physically remove particulate mass from the water which include flocculation and sedimentation with iron salts and alum, and filtration. Disinfection of the water most commonly relies on chemical treatment using chlorine, chlorine dioxide, or monochloramine which can remain in residual concentrations at the point-of-use and may result in biproductsSeveral small tap water studies have been performed on compatibility of tap water with CVD 103-HgR active component during the development of the PXVX0200, including waters from three US cities  and four European cities . CVD 103-HgR viability and water chemistry experiments were performed on tap water samples from San Diego, Redwood City, and Madrid  per the package insert instructions. Viability  was assessed immediately after reconstitution, and then at 15 and 30 min post-reconstitution. Water samples were tested in duplicate. Duplicates were performed by reconstituting the vaccine in 100 mL from two separate bottles of bottled water, or two separate 100 mL aliquots of tap water. Five samples from each reconstituted vaccine were plated for CFU analysis per time-point.5 CFU/dose. Colonies were quantified using the ProtoCOL 3 Plus automatic colony counting and zone measuring system (Synbiosis). Five TSA plates were inoculated using the spiral plater with 50 μL of sterile WFI quality water as a negative control for each experiment, with no more than 5 colonies/plate allowed.In all cases, reconstituted vaccine sample was diluted into chilled buffered peptone water (kept on ice) to achieve concentrations within the linear range of the CFU assay . Samples were plated onto tryptic soy agar (TSA) plates  using the WASP Touch Spiral Plater (Don Whitely Scientific) or Autoplate Spiral Plating System . Sample were diluted and plated using the 50 μL logarithmic mode, per plating system instructions. The theoretical lower limit of detection of the assay is 2 × 10Seven lots of CVD 103-HgR containing active component were used in these studies. Lot FA03 and lot 001 were produced in San Diego, and lots 054, 230, 231, 232, and 076 were produced in Bern, Switzerland. Buffer component lots were used interchangeably depending on availability.8 CFU/dose) after reconstitution in water.All stability time-points were determined using the CVD 103-HgR active product reconstituted in chilled buffered peptone water (Hardy Diagnostics) per the SOP for the PXVX0200 vaccine stability program. Stability is defined as the ability of Vaxchora to maintain a CFU/dose titer above the lower limit of potency (4 × 10An early study (July 2015) was performed to investigate the viability of CVD 103-HgR in tap waters from Miami (Florida), San Diego  and Redwood City . A second PXVX0200 tap water viability study was performed in May–June 2018 using tap waters that were obtained from Milan , London (UK), Bern (Switzerland), and Madrid (Spain) in Europe. Finally, fresh samples of tap water were collected in February–March 2019 from San Diego, Madrid, and Redwood City for re-testing using both CFU and water chemistry in order to correlate the effect of specific chemical compounds with the CVD 103-HgR active component viability. San Diego and Madrid tap water were selected because of observed effects on the viability of the CVD 103-HgR active component when used for reconstitution in the earlier studies, and Redwood City tap water was chosen as a control since CVD 103-HgR active component viability was not affected in the previous experiments. All samples were collected in 1L PETG bottles and shipped to San Diego where they were stored at 2–8 °C until testing.Chemical analysis panels were performed on aliquots of the San Diego, Madrid, and Redwood City tap water collected in February–March 2019 by EnviroMatrix Analytical, Inc in San Diego, CA. Tests included Total Metals by EPA 200 methods, Organochlorine Pesticides by EPA method 8081B, Organophosphorus Pesticides by EPA method 8141A, Volatile Organic Compounds by EPA method 8260B, and conventional chemistry parameters such as bicarbonate alkalinity and carbonate alkalinity by National Environmental Methods Index (NEMI) method 2320 B, ammonia as N (EPA method 350.1), chloride (NEMI method 4500-Cl B), residual chlorine and chloramines (NEMI method 4500-Cl G), specific conductance (NEMI method 2510B), methylene blue active substances (NEMI method 5540 C), total phenols (EPA method 420.1), and phosphorus . Additional testing was performed specifically for total organic carbon (NEMI method 5310B), fluoride (EPA method 300.0). Water chemistry methods were performed in singlicate.9, which was then used to reconstitute PXVX0200 buffer and active components. For example, the MCL for copper is 1.3 mg/L so for these studies copper was spiked into the MBGW to a create a solution with the final concentration of 13 mg/L copper. If the viability of CVD 103-HgR was affected at the 10X concentration, fresh samples were prepared at lower concentrations to determine the sensitivity of the CVD 103-HgR bacteria to the contaminant. 100 mL of the solution was used to reconstitute the vaccine, and samples were taken at 0, 15, and 30 min post reconstitution samples to test for CVD 103-HgR viability, as described above. The chemicals that were investigated were Bromodichloromethane , bromoform , dibromochloromethane , copper sulfate , and fluoride .Using the results of the water chemistry analysis, candidate chemical compounds were identified that could potentially affect the CVD 103-HgR active component viability. The chemicals were individually spiked into molecular biology grade water (MBGW) samples at levels that were 10-times the maximum contaminant level (MCL) allowed by the EPA regulationsA separate study was performed to investigate the viability of CVD 103-HgR active component when reconstituted in water spiked with sodium hypochlorite  at 5, 10, and 100 mg/L.16. European brands of still and sparkling were chosen based on data 83% of bottled waters produced in Europe are labelled Natural Mineral Water according to the European Federation of Bottled Waters, a trade association representing the interests of the European bottled water industry. Detailed market share data on the most popular brands of bottled water in the EU were not available, so brands were chosen based on availability from two international retail chains, REWE (Germany) and Carrefour (France), including the store brands, because of the availability between countries. Representative bottled waters from different regions of Europe were chosen to ensure more thorough analysis.Purified bottled and bottled spring water brands in the US were selected based on the sales of the leading bottled still water brands according to the 2015 Statista Bottled Water in the U.S. dossierPurified bottled water brands used for these studies were DASANI, Aquafina, Nestlé Pure Life, Glaceau Smartwater, Niagara, and Kirkland Signature. Bottled spring water brands used for these studies were Poland Spring, Deer Park, Ozarka, evian, Crystal Geyser, and Arrowhead.European brands of still Natural Mineral Water used for these studies were Volvic and Evian (France), Acqua Panna , Henniez (Switzerland), REWE (Germany) and Carrefour (France). European sparkling water brands used for these studies were S.Pellegrino , Gerolsteiner (Germany), San Benedetto and Ferrarelle , Radenska (Slovenia), and Perrier (France).8 CFU/dose for several possible sample sizes. In the simulations, the log10-transformed CFU data were assumed to be Gaussian, and the mean and standard deviation of the data-generating distribution were calculated using the results from a pilot study. We selected a sample size of 6 brands of each type of bottled water for the current study in order to achieve 99% power.Statistical analyses and power calculations were performed in the software R, v. 3.5.1 (R Core Team 2018) from IBM. Mean and standard deviation and the assumption of normality on the log scale were used to calculate the probability that the lower bound of the 95% confidence interval on the geometric mean of the CFU/dose at 15 min post-reconstitution across multiple types of bottled water would equal or exceed the lower acceptance criterion of 4 × 10Results from early PXVX0200 product development studies are shown in Table 8 CFU/dose) at time zero, immediately after reconstitution, which then decreased to below the level of quantification for the CFU assay at 15 and 30 min post-reconstitution. PXVX0200 reconstituted in tap waters from Miami and Redwood City remained above the potency specification for at least 30 min following reconstitution.Reconstitution of PXVX0200 in San Diego tap water resulted in CVD 103-HgR viability below the lower potency limit of the vaccine . The other replicate values for Madrid tap water 15 and 30 min post-reconstitution were within specification , so the averages for the duplicates were also within specification though the variability in the measurements was high (> 30% relative standard deviation) for these replicates.CVD 103-HgR viability was maintained in all tap water samples from Europe, however one of the duplicate measurements from the Madrid sample at 15 and 30 min post-reconstitution were below the specification , and Madrid were compared, and candidate chemical compounds were identified. We focused on chemical compounds that were by-products of the disinfection process, and chemical compounds found at higher concentration in San Diego and Redwood City tap water compared to Madrid. If the chemical compound was not found in any of the three samples, it was not included Table .Table 3WThe results from spiking the chemical compounds into MBGW are shown in Table The CVD 103-HgR active component reconstituted in MBGW spiked with 13 mg/L copper, which is 10 times the MCL allowed in drinking water, was not viable, nor was it viable in 0.078 mg/L MBGW spiked copper solution, which correlated to the lowest concentration of copper observed in the water chemistry panels (Redwood City).The CVD 103-HgR active component viability was not substantially affected by bromodichloromethane, chlorodibromomethane, bromoform, or fluoride spiked at 10 times the MCL allowed in drinking water.Chlorine was effectively neutralized by the ascorbic acid in the buffer component, though chlorine at 100 mg/L (25 times the MCL) appears to affect the viability.Bottled spring and purified bottled water studies Table  were perEuropean still bottled water experiments were performed in May 2019 with CVD 103-HgR active lot 230 and sparkling bottled water experiments were performed in April–July 2020 with lot 076  and natural spring water (EU), was performed for each time-point Table .Table 7GThe combined data for all brands of bottled spring water, purified bottled water, and bottled natural mineral water remain above the lower limit of specification for the PXVX0200 vaccine for at least 30 min.The aim of this study was to investigate the compatibility of CVD 103-HgR, the active ingredient in the PXVX0200 oral cholera vaccine, with drinking water from different sources in the US and Europe. Tap water quality in developed nations is regulated by law, however chemicals are still common and may be derived from natural sources, disinfection procedures, and plumbing.17. Further investigation into the specific chemistry of San Diego and Redwood City tap waters collected in February/March 2019 revealed several contaminants at detectable levels. Spiking studies were performed with these contaminants and it was found that that copper, even at relatively low concentration (0.078 mg/L) substantially affected the viability of CVD 103-HgR active component. The most common source of copper in drinking water is corrosion of plumbing, faucets, and water fixtures19. Based on these results, tap water should not be used to reconstitute the PXVX0200 cholera vaccine prior to administration.The CVD 103-HgR active component was not viable in San Diego tap water, and demonstrated various levels of viability when reconstituted in tap waters from Madrid and Redwood City. One possible cause of the variation is that the waters were collected in different years and during different months. The CVD 103-HgR active component was stable when reconstituted in tap water collected from Redwood City, California, in July 2015, but wasn’t stable when reconstituted in water from the same source collected in February, 2019. Conversely, the viability of CVD 103-HgR active component was inconsistent when reconstituted in tap water collected from Madrid in May/June 2018, but was stable when reconstituted in tap water from the same source collected in February/March 2019. This suggests that there is seasonal variability in tap-water chemistry, which is supported by the literatureAll bottled water brands tested from the US and European countries were compatible with the CVD 103-HgR active component, including 6 bottled spring and 6 purified bottled water brands in the US, and 12 natural mineral waters (6 still water brands and 6 sparkling water brands) from Europe. The PXVX0200 reconstitution instructions include only bottled spring and purified bottled waters in the US, and still bottled waters in Europe.These results support the conclusion that tap water should not be used to reconstitute the PXVX0200 vaccine, but instead still bottled spring, purified bottled, and natural mineral waters may be used.


Dietary calcium is essential for optimal egg production and quality in laying hens, but high concentrations can impede the digestibility of other dietary components. The provision of limestone grit in addition to the main diet may help maintain overall calcium intake while allowing a reduction in dietary calcium levels. The impact of phytase, an enzyme that increases calcium availability in the gut, on the voluntary consumption of limestone grit is unknown. Here, the capacity for hens with access to a separate limestone grit source to modify Ca consumption when offered varying dietary Ca levels and phytase was evaluated. Dietary phytase reduced limestone grit consumption. Egg production was unaffected by reducing dietary calcium levels or the phytase addition. Eggshell measurements worsened in line with decreasing dietary calcium levels despite the provision of limestone grit. In summary, the provision of limestone grit resulted in comparable egg production but not eggshell quality in hens offered suboptimal levels of dietary calcium. p = 0.024). Egg weight, rate of lay, and egg mass were unaffected by dietary treatment (p > 0.05). Egg shell weight % (p < 0.001), shell thickness (p < 0.001), and shell breaking strength (p < 0.01) decreased in line with dietary Ca levels. In summary, dietary superdosing with phytase reduced the consumption of a separate limestone source in individually housed, early lay ISA Brown hens. Egg shell quality variables but not egg production worsened in line with lower dietary Ca levels. Laying hens require substantial quantities of calcium (Ca) to maintain egg production. However, maintaining recommended dietary Ca through inclusion of limestone may impede nutrient digestibility, including that of other minerals. It was hypothesized that providing a separate source of dietary Ca in the form of limestone grit would preserve Ca intake of hens offered diets containing suboptimal Ca concentrations. Furthermore, the impact of dietary phytase at a “superdosing” inclusion rate on the voluntary consumption of limestone grit was evaluated. One hundred and forty-four laying hens (19 weeks of age) were assigned to one of six dietary treatments in a 3 × 2 factorial arrangement comprising three dietary Ca concentrations  and ±dietary phytase (3500 FYT/kg diet) on an ad libitum basis for six weeks. Limestone grit (3.4 ± 1.0 mm) was provided to all hens ad libitum. Hens offered diets containing phytase consumed significantly less limestone grit  Laying hens have a large requirement for dietary Ca to satisfy eggshell synthesis, and this is typically satisfied by dietary inclusion of limestone and other various calcium-containing ingredients. However, dietary limestone is associated with an increase in the pH of avian gastrointestinal tract contents ,2 which Providing a choice of diets differing in nutrient profile is a dietary strategy that allows birds to select nutrients based on individual requirements. Holcombe et al.  reportedThe use of phytase to enhance the availability of phytate-bound minerals including Ca has been widely embraced in the broiler sector, although evidence for improvements in laying hen egg production is lacking and conflicting ,14. NoneTherefore, the objective of this study was to evaluate the choice of feed and limestone grit intake, as well as the impact on egg quantity and quality characteristics of hens offered diets with varying dietary Ca concentrations and the inclusion of phytase.The experimental procedures that were conducted in this study were approved by the University of Sydney Animal Ethics Committee (Project Number 2016/945) and were conducted in accordance with the Australian code for the care and use of animals for scientific purposes [A total of 144 ISA Brown hens (17 weeks of age) were housed individually within their cages measuring 25 × 50 × 50 cm with three adjacent cages forming a statistical unit. Hens were habituated to the cages and offered a common point of lay ration for two weeks before experimental treatments were assigned and measurements commenced (19 to 24 weeks of age). At 19 weeks, the rate of lay was 90%. The photoperiod regimen was 16 h of light and 8 h of dark. Each trio of adjacent hens were randomly assigned to one of the six treatment groups, allowing for 8 replicates/treatment spaced randomly throughout the layer house. Diets based primarily on wheat, sorghum, and soybean meal and differing only in dietary Ca  and the inclusion or exclusion of phytase  were offered in mash form on an ad libitum basis for a duration of 6 weeks. The ingredient and nutrient compositions of the layer diets are shown in During the experimental period, egg quality analysis was conducted once weekly shortly after egg collection on the same day each week. Eggs were allowed to reach room temperature in the laboratory prior to the onset of measurements. Both internal and external egg quality parameters were measured and recorded. Egg weight (g), height (mm), and width (mm) were measured, using a digital scale for weight, to an accuracy of 0.01 g, and a digital caliper  for height and width. Egg height was measured as the length from pole to pole, while width was measured at the equator. Egg breaking force (peak force) was measured using a texture analyzer . For internal egg quality testing, the breakout method was employed, using a flat, levelled glass surface on a metal stand with a reflective mirror. Yolk color was determined using a DSM Yolk Colour Fan , and assigned a value from 1 to 15 units. Using a digital caliper, albumen and yolk width were measured at their widest distance at a 90° angle to each other. Albumen height was measured using a QCD AH reader . Yolk height was measured using a digital height gauge . Albumen and yolk were then separated using a plastic spatula and placed in weigh boats for weighing (g) purposes. Haugh values (1) were calculated using the formula: in grams . Eggshell weight was estimated by gently wiping residue albumen from shells with a paper towel and then air-drying for three days at room temperature. Once dried, eggshell weight was measured using digital scales to an accuracy of 0.01 g. Egg shell thickness was measured at three egg segments  using a digital caliper  and then averaged. Total excreta were quantitatively collected from each cage, and feed intake was recorded for a 72 h collection period to determine the apparent metabolizable energy (AME) on a dry matter basis, nitrogen (N) retention, and N corrected AME (AMEn). Diets, limestone supplement, and excreta were dried in a forced-air oven at 80 ℃ for 24 h, and the gross energy (GE) of feed and excreta output was determined using a Parr 1281 adiabatic bomb calorimeter  which was standardized with benzoic acid and used to determine AME (2) using the following calculation: The N content of diets and excreta was determined using an elemental analyzer , and N retention (3) was calculated from the following equation:AMEn (MJ/kg) values were calculated by correcting to zero N retention, using the factor of 36.54 kJ/g N retained in the body . The retThe mineral composition of the feed, excreta, and limestone grit was determined by inductively coupled plasma optical emission spectrometry (ICP) using a PerkinElmer OPTIMA 7300  following digestion with nitric acid and hydrogen peroxide beforehand. The coefficient of Ca retention for combined feed and limestone grit usage and for other feed minerals was calculated using the equations above. p < 0.05, <0.01, and <0.001. The development of limestone grit usage, egg weight, rate of lay, and feed intake over time was assessed by repeated measures using the R studio software  with the package “rstatix” [The data were analyzed as a 2 × 3 factorial arrangement of treatments using the GLM procedure of SAS . The starstatix” . LimestoThe dietary ingredients and calculated and analyzed composition are presented in p = 0.004). Daily feed intake did not differ between treatment groups. However, there was an interaction between phytase and dietary Ca concentrations on calculated daily Ca intake from feed. Daily Ca intake from feed was significantly greater in the 20 g/kg dietary Ca treatment groups supplemented with phytase when compared with complementary Ca diets containing no phytase (p = 0.007). However, there was no effect of phytase on daily Ca intake from the 40/g/kg and 30 g/kg dietary Ca group. These differences reflect the measured differences in dietary Ca between phytase inclusion at each Ca level. The overall response of limestone grit intake to dietary treatments is presented in p = 0.024). This effect was the most pronounced in the 40 g/kg Ca diets. The combined intake of Ca from the diet and the limestone grit was affected by dietary Ca level, whereby hens offered the 40 g/kg Ca diets had the greatest total Ca intake, followed by those of the 30 g/kg Ca diets and then the 20 g/kg Ca diets. Phytase inclusion had no effect on total Ca intake. Egg weight, egg mass, and FCR were unaffected by dietary treatments. Overall rate of lay was unaffected by dietary treatment. However, when assessed on a weekly basis, the effect of phytase on laying performance differed among Ca intake groups, where hens which were fed with 40 g/kg Ca and no phytase experienced a higher laying rate  and compared . Initialp = 0.030, p = 0.010, and p = 0.001, respectively). Similarly, the yolk color and eggshell breaking force was the greatest for eggs obtained from hens fed the 40 g/kg Ca diets and statistically significantly different when being compared to those of eggs obtained from hens fed the 20 g/kg Ca diets . Shell thickness was the greatest for eggs obtained from hens fed the 40 g/kg Ca diets, intermediate and significantly different for the eggs obtained from hens fed 30 g/kg Ca diets, and thinnest for the eggs obtained from hens fed 20 g/kg Ca diets . The response of egg quality measurements to dietary treatments is presented in p = 0.084). The addition of phytase to the 40 g/kg dietary Ca increased the coefficient of dry matter retention by 14%. This response to phytase was not observed in the 30 g/kg and 20 g/kg Ca diets. There was an interaction between dietary Ca concentrations and phytase inclusion on Ca retention. Ca retention was lower in the 30 g/kg Ca diet containing phytase when compared with that in the 30 g/kg Ca diet without phytase. The retention of Ca was greater but not significantly different in the 40 g/kg Ca diet containing phytase when compared to that of the same Ca concentration diet without phytase. There was no effect of phytase on Ca retention on hens offered the 20 g/kg Ca diets. There was an interaction between phytase inclusion and dietary Ca concentrations on Cu retention. The retention of Cu was greater for the hens offered the 30 g/kg Ca diet and phytase when compared with that of hens fed the 30 g/kg Ca diet without phytase. However, there was no effect of phytase supplementation on Cu retention at other dietary Ca concentrations. There was an interaction between dietary Ca concentrations and phytase for K retention. K retention was higher for birds offered the 40 g/kg Ca diet and phytase when compared with the that of birds fed the same Ca concentration diet containing no phytase. However, there was no effect of phytase supplementation on K retention of hens offered the 30 g/kg and 20 g/kg Ca diets. Finally, there was a significant effect of dietary Ca concentrations on Fe retention. Fe retention was the greatest for the 20 g/kg Ca diet, intermediate and not different for the 40 g/kg Ca diet, and lowest for the 30 g/kg Ca diet (p < 0.05). The response of measurements of digestibility and nutrient retention are presented in A hypothesis of this study was that reducing dietary Ca and providing supplementary limestone grit may preserve overall Ca intake but with some additional benefits in performance and nutrient retention. The guidelines around Ca nutrition for ISA Brown hens in early lay suggests an ideal concentration of 40–41 g Ca/kg diet with an expected feed intake of between 114–115 g/hen/day, giving an estimated Ca intake of approximately 4.6 g of Ca/hen/day . In thisEgg production variables such as egg weight, egg mass, rate of lay, and FCR were unaffected by dietary treatments. This suggests that the supplementary limestone consumed in the lower Ca diets preserved these variables. However, caution needs to be exercised in speculating here, as the study focused on early lay birds and only for a six-week period. The hens assigned to the 20 g/kg Ca diets had a lower final body weight. This suggests that these hens were possibly compensating for the undersupply of dietary Ca relative to the other Ca diet groups. Similarly, Ziaei et al.  reportedEgg quality variables and particularly those related to shell strength and weight were negatively impacted in birds offered the 20 g/kg Ca diets when compared to those of eggs obtained from hens fed the 40 g/kg Ca diets and, occasionally, eggs obtained from hens fed the 30 g/kg Ca diet. This indicates that even with supplementary limestone grit provision, maintaining adequate Ca intake from the basal diet is still an important objective, particularly when the variability in limestone grit consumption between hens as observed in this experiment is considered. While there was an impact of superdosing with phytase on limestone grit consumption, particularly in the 40 g/kg Ca diet, there was no impact of adding the enzyme on hen body weight gain or any other investigated production parameters. In this study, the levels of non-phytate phosphorus were replete for ISA Browns in early lay, and this may explain the absence of an effect of phytase in improving production parameters. However, Kim et al.,  reportedThe inclusion of phytase significantly increased Ca retention in the 40 g/kg Ca diets from 0.529 to 0.740. However, it reduced or did not affect Ca retention in the 30 and 20 g/kg Ca diets. A similar outcome was observed for the retention of dietary K. These interactions are difficult to account for, but nonetheless raise some interesting questions into how high Ca intake and greater Ca bioavailability as effected by phytase may affect voluntary usage of a supplementary limestone grit in laying hens. The review of Selle et al. [In conclusion, egg production variables were not impacted by lowering dietary Ca level or the addition of phytase in early lay hens offered an additional limestone source. The results of the present study showed that overall limestone grit consumption was not uniform across laying hens. Hence, hens fed the 20 and 30 g/kg Ca diet were not able to compensate Ca intake adequately using the supplementary limestone, resulting in inferior egg quality. In addition, the inclusion of phytase did not impact any investigated performance parameters but decreased limestone intake in hens fed the 40 g/kg Ca diet. Hens can benefit from limestone grit supply when being fed a 40 g/kg Ca diet which will help them to maintain their laying performance, egg quality, and production.


Proteobacteria, and norepinephrine were positively correlated with Bacteroidetes, but negatively associated with Firmicutes phylum. The present study demonstrated alteration in the gut microbiome between persons with IBS and emotional distress compared to HC. The correlations between plasma neurotransmitters and the gut microbiome suggest that the gut microbiome may impact the regulation of neurotransmitters.Evidence highlights the comorbidity between emotional distress and irritable bowel syndrome (IBS) through the gut-brain axis. However, the underlying mechanism is largely unknown. Thus, the present study aimed to evaluate the associations among neurotransmitter levels and the gut microbiome profiles in persons with IBS and emotional distress. In this nested case-controlled study, emotional symptoms, including anxiety and depressive symptoms, were evaluated in 40 persons with IBS and 20 healthy controls (HC). Plasma neurotransmitters levels (serotonin and norepinephrine) and the gut microbiome profile of the collected fecal samples were examined. Emotional distress and microbiome profile were significantly different between IBS and HC groups. Lower but not significant neurotransmitters’ levels (serotonin and norepinephrine) were observed in the IBS group compared to the HC. A negative correlation was found between norepinephrine levels and alpha diversity (Shannon and Simpson indices) in the IBS group. Moreover, serotonin levels were positively associated with the abundance of  IBS affects 10–15% of adults worldwide and is associated with a broad spectrum of emotional distress comorbidities4. Epidemiological data report that 40–90% of persons with IBS experience some degree of anxiety and depressive symptoms in their life6. Emotional distress may influence IBS symptom perception and illness behavior in patients, contribute to poor quality of life as well as increase health care costs9. A comorbid condition may suggest a biological association between emotional distress and IBS5.Irritable bowel syndrome (IBS), the most common functional gastrointestinal disorder, is characterized by chronic abdominal pain and alteration in bowel habits11. This association may be related to the bidirectional communication between the central nervous system (CNS) and the gut neuroendocrine system known as the brain-gut axis12. Emotional distress may lead to homeostasis shifts along the brain-gut axis and alteration in the gut microbiome pattern in IBS13. Several studies reported similarities in the fecal microbiome of persons with IBS and persons with depression , which highlights the bidirectional interaction between the brain and the gut in comorbidity between emotional distress and IBS14.The relationship between IBS and emotional distress is supported by neuroimaging and psychophysiological studies16. It has been postulated that insufficient levels of the monoamine neurotransmitters  may lead to depression17. Serotonin, as a key neurotransmitter of the brain-gut axis, plays an important role in the pathogenesis of both emotional distress and IBS6. More than 90% of the body’s serotonin is synthesized in the gut19. Various bacteria such as Streptococcus spp., Enterococcus spp., Escherichia spp., Lactobacillus plantarum, Klebsiella pneumonia, and Morganella morganii have the ability to produce serotonin23. The literature also supports that other neurotransmitters such as dopamine and norepinephrine can be produced by bacteria such as Lactobacillus, Serratia, Bacillus, Morganella and Klebsiella. Interestingly, dopamine and norepinephrine can stimulate the growth of E. coli O157: H725. Alteration of the gut microbiome may change the biosynthesis, release, and reuptake of neurotransmitters. The alteration may result in a disturbance in processes operating between the CNS and the enteric nervous system and may lead to emotional distress26. Described mutual interaction between the CNS and the gut highlights the role of the gut microbiome and neurotransmitters in emotional distress27.Neurotransmitter imbalance is one of the mechanisms attributed to emotional distress28. Moreover, the gut microbiome as a cardinal part of the gut-brain axis has gained significant interest in the pathophysiology of both IBS and emotional distress in recent years. Thus, in the current study, we hypothesized a correlation between the gut microbiome and plasma neurotransmitters levels in IBS people with emotional distress. We aimed to investigate the difference in biomarkers (neurotransmitters levels and gut microbiome diversity) between IBS and healthy controls (HC), as well as the relationship between the gut microbiome and neurotransmitters in persons with IBS and emotional distress.Traditionally, emotional distress such as depression has been related to an imbalance in serotonin, norepinephrine, and dopamine levels. Some studies evaluated the role of serotonin in IBS, but research about the function of other involved neurotransmitters such as norepinephrine is limitedDemographic information of the participants, including gender, age, race, ethnicity, and marital status, is shown in Table The mean anxiety scores were 62.85 ± 7.67 and 54.41 ± 6.12 in the IBS and HC groups, respectively. Moreover, the mean of depressive symptoms were 56.11 ± 7.30 and 48.55 ± 6.26 in the IBS and HC groups correspondingly. The results showed that the mean of anxiety and depressive symptoms were significantly higher in the IBS group compared to the HC group. While the HC anxiety and depressive symptoms were within the normal limits, the IBS group had moderate anxiety level and mild depressive symptoms. The results can be found in Table The mean plasma serotonin level was 35.47 ± 27.53 ng/ml and 42.36 ± 31.94 ng/ml and the norepinephrine level was 358.66 ± 120.25 pg/ml and 395.57 ± 169.84 pg/ml in the IBS and HC groups, respectively. While plasma serotonin and norepinephrine levels were lower in the IBS group, they were not significantly different compared to the HC group. The results are shown in Table Fusobacteriaceae, Fusobacteria, Fusobacteriales and Fusobacales in the HC group and a high abundance of Lactobacillaceae, Lactobacillus, Flavonifractor, Lactobacillales, Bacilli, Coriobacteriales, Coriobacteriia, Coriobacteriaceae, Erysipelotrichia, Erysipelotrichaceae, Erysipelotrichales, Oscillibacter, Blautia, Burkholderiales, Betaproteobacteria, Alcaligenaceae, Parabacteroides and Porphyromonadaceae in perspons with IBS compared to HC , Simpson (p = 0.019), and Shannon (p = 0.013)  was measured using three different alpha diversity indices, including total observed species (sobs), Simpson , and Shannon  indices. According to these indices, there was an increased bacteria alpha diversity in the IBS group compared to the HC group (sobs 3) Figs. , 3, 4.Fi Figs. , Moreover, there was significant dissimilarity in beta diversity  using Bray–Curtis (abundance or read count data) and Jaccard (the presence/absence of species) dissimilarities between the two groups Figs. , 6. The p = 0.029 and p = 0.015; respectively)  Table .Table 4Proteobacteria (p = 0.021), a significant negative correlation between norepinephrine and Firmicutes (p = 0.020), and a significant positive correlation between norepinephrine and Bacteroidetes phylum (p = 0.043)  Table .Table 5CThe current study assessed the difference in the plasma neurotransmitter levels and the gut microbiome profile in persons with IBS who experienced anxiety and depressive symptoms compared to the HC. Also, we evaluated the correlation between neurotransmitter levels and the gut microbiome diversities and microbial abundance at the phylum level. Our findings revealed that gut microbiome patterns in persons with IBS who suffer from emotional distress are different from HC. Although there was no difference in neurotransmitter levels between the two groups, we observed a correlation between bacterial profile  and neurotransmitters’ levels in the IBS group.29. Thus, high comorbidity between IBS and emotional distress, including anxiety and depressive symptoms, may be a consequence of an alteration in serotonin and norepinephrine31. Both the CNS and the enteric nervous system have the capability to produce neurotransmitters28. A decrease in the amount of serotonin and norepinephrine released and deterioration of neurotransmitters binding to receptors may lead to emotional distress32. In the current study, no significant difference was observed in either serotonin or norepinephrine levels between IBS and HC. Our results are in contrast to the findings of Zhao et al. (2014) in which IBS patients with depressive symptoms had lower plasma serotonin33. While serotonin has drawn a tremendous amount of attention regarding the pathogenesis of comorbidity of depressive symptoms and IBS, some studies have reported a lack of significant correlation between plasma serotonin levels and depressive symptoms in IBS patients compared to healthy controls36. Although alteration in serotonin levels has been highlighted in the comorbidity of IBS and depressive symptoms33, there are limited studies to investigate the role of other neurotransmitters such as norepinephrine in comorbidity between IBS and emotional distress. Thus, further research is needed to assess the role of norepinephrine in this comorbidity.It has been hypothesized that the imbalance in monoamine neurotransmitter levels  is a key factor in the pathophysiology of depressive symptoms and anxiety disorders18. The gut microbiome may influence basal circuits of emotion processing via the vagal pathway from the gut38. Interoceptive stimuli may impede conscious and subconscious emotional and cognitive processing39. Research has shown that the dysbiosis in the gut microbiome is a shared abnormality in the pathophysiology of both IBS and depressive symptoms40. We observed higher alpha diversity in terms of total observed species, Simpson and Shannon indices in persons with IBS compared to HC. Our results contrast with the findings of Peter (2018), who reported non-significant lower alpha diversity in IBS patients with psychological distress13. Similarly, Hugerth (2019) found no association between bacterial diversity and depressive symptoms in IBS patients41. While we observed higher alpha diversity, Liu (2016) reported low alpha diversity (Shannon index) in persons with IBS and depressive symptoms14. Sundin (2015) also showed a negative correlation between fecal microbial diversity and anxiety and depressive symptoms in patients with IBS42. While pre-clinical studies have supported the hypothesis of lower alpha diversity in animal models after exposure to stress44, the results in clinical studies are contradicted and require further investigations. Regarding beta diversity, similar to our findings, Peter (2018) found that microbial beta diversity was associated with depressive symptoms13. Beta diversity as an indicator of taxa similarity within samples may clarify how emotional distress segregated the gut microbiome features among different subgroups of individuals that require further investigation45.It has been postulated that the gut microbiome contributes to the pathogenesis of IBS and emotional distress, such as depressive symptoms and anxietyBacteroidetes, Firmicutes, and Verrucomicrobia in persons with IBS who suffer from emotional distress. Similar to our findings, elevated Lachnospiraceae were reported in cases of both anxiety and depressive symptoms13. However, studies have shown mixed results concerning the abundance of various gut microbiomes at different taxonomic levels among persons with IBS who had emotional distress, and thus, additional studies are needed46.We also found a high abundance of several bacteria at different taxonomic levels that belong primarily to the three phyla Proteobacteria phylum as well as norepinephrine and Bacteroidetes phylum. However, Firmicutes had a negative association with norepinephrine.Recent evidence has highlighted the ability of the gut microbiome to produce neurotransmitters, which may lead to the development/maintenance of emotional distress such as anxiety and depression. While we did not observe any association between various alpha diversity indices and serotonin, there was a negative correlation between alpha diversity (Shannon and Simpson indices) and norepinephrine levels. Moreover, there was a positive association between serotonin and 19. For instance, Candida, Escherichia coli, Streptococcus, and Enterococcus may secrete serotonin and Escherichia coli, Bacillus and Saccharomyces can produce norepinephrine48. The production of neurotransmitters by various microbiota may lead to the imbalance of neurotransmitter levels and the development of pathogenic bacteria. For example, the presence of norepinephrine may stimulate the growth of Escherichia coli25. Additionally, studies show that the in vitro exposure of the Klebsiella pneumoniae, Enterobacter cloacae, Pseudomonas aeruginosa, Shigella sonnei, and Staphylococcus aureus to norepinephrine may lead to the growth of these pathogens49. So, alteration of the gut microbiome due to IBS may be attributed to the imbalance in neurotransmitters levels and subsequently emotional distress. Accumulated preclinical studies highlight the role of the gut microbiome in regulating neurotransmitters and depressive-like behavior in the animal models50. There is a strong need for future clinical studies to understand the biological mechanism of comorbidity of emotional distress with IBS by shedding light on the role of the gut microbiome in the production of neurotransmitters.Research shows that more than 90% of the serotonin in the human body is synthesized by the gutThere are limitations in the study that need to be considered when interpreting the results. The small sample did not allow the categorization of persons with IBS based on the severity of the emotional distress. Therefore, the interpretation of the biomarkers based on the severity of emotional distress is limited. Moreover, a small sample size and unbalanced design might increase the likelihood of a type II error skewing the results. Therefore, caution is needed when interpreting the results of the study, which needs to be replicated in a larger sample. The majority of the participants in our study were mixed type (IBS-M) according to the self-reported symptoms. Further studies with a larger sample size by considering history of diet, IBS subtype and other IBS symptoms  are recommended to determine the associations between neurotransmitters and the gut microbiome in persons with IBS and emotional distress.The study’s findings highlight that persons with IBS and emotional distress may be differentiated from healthy persons based on their gut microbiome profile. There were lower but not significant neurotransmitter levels in the IBS group compared to the HC. Also, we found correlation between the gut microbiome  and neurotransmitters in persons with IBS and emotional distress. Future research in a larger sample is required to correlate neurotransmitter levels with observed gut microbiome alterations. Identification of biomarkers may help advance IBS management with comorbid emotional distress.NCT03332537)51. In the parent study, 80 persons with IBS diagnosed by a gastroenterologist were enrolled from the general community as well as two large public university campuses and two gastrointestinal (GI) clinics in the northeastern region of the United States. The participants completed emotional distress questionnaires, and their blood and fecal samples were collected at three different time points: baseline, six weeks, and 12 weeks. The data from the parent study's baseline session was utilized and 20 healthy subjects were recruited as controls in the present study.The present cross-sectional comparative study is part of a parent randomized clinical trial titled “Precision Pain Self-Management in Young Adults with IBS” (52.The inclusion criteria for enrollment of people with IBS in the published protocol of the parent study were: (1) men and women 18–29 years of age, (2) with a diagnosis of IBS from a healthcare provider using the Rome IV criteria, and (3) able to read and speak in English. The exclusion criteria were: (1) having other chronic painful conditions including but not limited to fibromyalgia, chronic pelvic pain or chronic intestinal cystitis, infectious diseases , celiac disease or inflammatory bowel disease, and/or diabetes mellitus, (2) serious mental health conditions , (3) women pregnant or post-partum within 3 months of delivery, or, (4) regular use of opioids, iron supplements, prebiotics/probiotics or antibiotics, and/or substance use. The criteria for recruitment of HC were the same as those for the IBS group, except that the HC group did not have a history of IBS or any chronic diseaseg for 20 min at 4 °C, and then plasma was aliquoted into eppendorf tubes and stored at − 80 °C until further analysis.The IBS and HC groups completed questionnaires via a Research Electronic Data Capture (REDCap) software/system. Peripheral blood samples were collected into EDTA tubes using a standardized venipuncture procedure. The collected blood samples were diluted using 2% fetal bovine serum and centrifuged at 1000×The participants were requested to collect their fecal samples at the time of enrollment in the study using a stool sample collection kit  per manufacturer instructions. Samples were mailed back to the research laboratory and frozen and maintained at − 80 °C until processing and 16S rRNA gene sequencing analysis.54.Anxiety and depressive symptoms were assessed using the Patient-Reported Outcomes Measurement Information System (PROMIS) v1.0 questionnaires from the recommended National Institute of Nursing Research (NINR) common data elements. PROMIS instruments measure clinically important outcomes, including physical, mental, and social health. PROMIS is scored by using the Health Measures Scoring Service55.The PROMIS questionnaire, the “Emotional Distress-Anxiety” section, which includes 6 questions, was used for measuring anxiety. Responders selected answers based on the category provided by the questionnaire . The researcher scored the responder's answers . After calculating the raw score by summing responses, the total raw score was translated to a T-score which is a standardized T-score with a mean of 50 and a standard deviation of 10. According to the PROMIS measurements, a score below 55 is within the normal limit, the score of 55–60 refers to mild, and a score above 60 indicates moderate and severe anxiety symptoms55.For measuring depressive symptoms, the PROMIS questionnaire, “Emotional Distress-Depression” section, which includes questions with five different answer categories  was used. The same approach was employed to calculate depressive symptoms according to the PROMIS measurement guidelines56. For this purpose, the steps mentioned in the guideline of Serotonin ELISA Assay Kit, including preparation of reagents , acylation of samples (20 µl) and ELISA test procedures were followed as per the manufacturer's instructions. The competitive Serotonin ELISA Assay kit uses the microtiter plate format, in which serotonin is bound to the solid phase of the plate. Antirabbit/peroxidase detects the antibody bound to the solid phase serotonin. The level of serotonin in the sample is inversely proportional to the amount of antibody bound to the solid phase serotonin. The sensitivity of the kit was 0.293 ng/ml and the range of detection was 0.49–500 ng/ml. The samples were run in duplicate, per batch and the mean inter- and intra-assay coefficients of variation for control samples was ≤ 20%.This study used the serotonin enzyme-linked immunosorbent assay (ELISA) assay kit named ADI-900-175  for the quantitative determination of serotonin in plasma57. For this purpose, 300 µl of plasma was processed according to the guideline of Norepinephrine ELISA Assay Kit, including preparation of reagents , preparation of samples, and ELISA test procedure per manufacture’s directions. The kit's sensitivity was 5 pg/ml and the range of detection was 0–32 pg/ml. The samples were run in duplicate, per batch and the mean inter- and intra-assay coefficients of variation for control samples was ≤ 20%.For the quantitative determination of norepinephrine in plasma, the norepinephrine ELISA assay kit named KA1891  was used58. Then the V4 region of the 16S rRNA genes of the microbial community were sequenced using the MiSeq Series Illumina platform.The fecal samples DNA extraction, sequencing, and analysis were carried out at the University of Connecticut, Center of Microbial Analysis, Resources, and Services (UConn MARS). Bacterial DNA was extracted from 0.25 g of stool sample using the MoBio PowerSoil isolation kit  in line with the manufacturer’s instruction for the Eppendorf epMotion 5076 Vac liquid handling robotThe sequences produced by PCR were normalized based on the concentration of DNA in the 350–400 bp region and pooled using the QIAgility liquid handling robot (Qiagen). Then, the pooled PCR was purified using GeneRead Size Selection kit  and paired-end sequencing (2 × 250) was performed on an Illumina MiSeq system.59 following the analysis pipeline of Miseq SOP (http://www.mothur.org/wiki/MiSeq_SOP) and the UConn MARS mothur batch file (https://github.com/krmaas/bioinformatics/blob/master/mothur.bash). Paired-end sequences were combined into contigs and aligned against to the SILVA 119 V4 16 S rRNA gene reference alignment database60. Operational taxonomic units (OTUs) were determined at a 97% identify, and then performing de novo OTU clustering on reads that failed to cluster to a reference. Taxonomic annotation was also determined by the SILVA 16S rRNA reference dataset. Sample were removed if the yielded reads were less than 10,000. For the microbiome analysis, the Mothur software59 was used. Alpha diversity, including Simpson, Shannon, and total observed species (sobs) indices, were used to evaluate the complexity of the whole microbial community. Beta diversity represented by Bray–Curtis and Jaccard was used to indicate the inter-subjects’ dissimilarity in the bacterial communities.The raw sequence data were processed by Mothur 1.42.3 Pipeline61. For comparing alpha diversity between the IBS and HC groups, a Wilcoxon rank-sum test was used. Also, based on Bray–Curtis and Jaccard dissimilarity, non-metric multidimensional scaling (NMDS) ordination for beta diversity was performed and multivariate dispersions (PERMDIST) test was conducted to examine the assumption of homogeneity. Spearman rank correlation was used to examine the association between neurotransmitter levels and alpha diversity indices  as well as bacterial phylum in the IBS group.For data analysis, the SPSS  version 26 and R version 4.0.2 were used. The demographic characteristics were presented with mean and standard deviation for continuous variables and frequency and percentage for continuous variables. The statistical approaches to assess the difference in emotional distress, and neurotransmitter levels between IBS and HC groups were Independent Sample T-test and Mann–Whitney. For analysis of microbiota composition, the linear discriminant analysis effect size (LEfSe) method was performedThe study was approved by the Institutional Review Board of the University of Connecticut and was conducted in accordance with the ethical guidelines of the Declaration of Helsinki. All the participants provided written informed consent.


Erythrinus erythrinus. We hypothesized that the presence of multiple sex chromosomes has contributed to the genetic differentiation of populations, which could have potentially accelerated speciation.Fish present astonishing diversity, comprising more species than the combined total of all other vertebrates. Here, we integrated cytogenetic and genomic data to investigate how the evolution of multiple sex chromosomes together with allopatry is linked to genetic diversity and speciation in the fish species Erythrinus erythrinus  being a notable example. This nominal species shows remarkable intra-specific variation, with extensive karyotype diversity found among populations in terms of different diploid chromosome numbers (2n), karyotype compositions and sex chromosome systems. Here, we analyzed three distinct populations  that differed in terms of their chromosomal features (termed karyomorphs) and by the presence or absence of heteromorphic sex chromosomes. We combined cytogenetics with genomic approaches to investigate how the evolution of multiple sex chromosomes together with allopatry is linked to genetic diversity and speciation. The results indicated the presence of high genetic differentiation among populations both from cytogenetic and genomic aspects, with long-distance allopatry potentially being the main agent of genetic divergence. One population showed a neo-X1X2Y sexual chromosome system and we hypothesize that this system is associated with enhanced inter-population genetic differentiation which could have potentially accelerated speciation compared to the effect of allopatry alone.Diversity found in Neotropical freshwater fish is remarkable. It can even hinder a proper delimitation of many species, with the wolf fish  Hoplias (13), Hoplerythrinus (3), and Erythrinus (2) [The biodiversity of Neotropical freshwater fish is remarkable, as they comprise at least 5200 species . The EryHoplias 1, Hopleryinus (2) , as all E. erythrinus and H. malabaricus, in which many studies have shown the presence of intraspecific variants, with extensive differences observed in the diploid chromosome numbers (2n), karyotype compositions and different sex chromosome systems among populations [E. erythrinus  with a prevalence of acrocentric chromosomes which distinguish the species karyotypes from those of other erythrinids [n = 54 chromosomes in both males and females, without the presence of differentiated sex chromosomes, karyomorphs B, C and D display a multiple ♀X1X1X2X2/♂X1X2Y sex chromosome system, in which males and females have 2n = 51 and 2n = 52, respectively [Such cryptic diversity is at least partially explained by the high karyotypic diversity found in some species, such as ulations . Four dithrinids ,6. Whileectively ,7.Even though allopatry is generally considered the main factor for the emergence of reproductive isolation and the reduction of gene flow that culminates in the speciation process , the impE. erythrinus  and used genomic approaches to investigate how the evolution of multiple sex chromosomes together with allopatry is linked to genetic diversity and speciation. We hypothesize that the presence of multiple sex chromosomes has differentially contributed to the genetic differentiation of populations, which could have potentially accelerated speciation.Recently, high-throughput sequencing technologies have opened a new era in population genetics, as they allow the assessment of complex demographic scenarios, including how gene flow and introgression affect genetic diversity and speciation . In thisThe collection sites, number and sex of the specimens investigated are presented in Here we present the first chromosomal data for population A2. Mitotic chromosomes were obtained by the protocol previously described . The 5S 0t-1 DNA ratio based on previous studies [0t-1 DNA, obtained from each population according to a previous study [0t-1 DNA from the respective population were co-precipitated in 96% ethanol, and the dry pellets were resuspended in 20 µL of hybridization buffer containing 50% formamide, 2 × SSC, 10% dextran sulphate and Denhardt’s buffer (pH 7.0). FISH was conducted under high stringency conditions, as described in [Comparative genomic hybridization (CGH) experiments were performed as described  with ada studies ,29,30. M studies  and labeus study , was incribed in .To confirm the diploid number, karyotype structure and hybridization results, at least 30 metaphase spreads were analyzed per individual. The microscopy images were captured using an Olympus BX50 epifluorescence microscope  coupled with a CoolSNAP camera, and the images were processed using Image-Pro Plus 4.1 Software . Final images were optimized and arranged using Adobe Photoshop, version CC 2020. Chromosomes were classified as metacentric (m), submetacentric (sm), subtelocentric (st) or acrocentric (a), according to their arm ratios . As the The DArTseq sequencing procedures were performed using liver tissues that were shipped to Diversity Arrays Technology Pty Ltd. . This method involves a complexity reduction in which the DNA is treated with PstI and SbfI enzymes before sequencing, which might enrich for sequences in lightly methylated regions . SequencA BayeScan analysis was carried out to test for markers with uncommon levels of inter-population differentiation (low or high), which might indicate locally adaptive alleles at the sequenced loci . We perfdH), Tajima’s D (D) and two measures of nucleotide diversity per site, π and Watterson’s theta (Wθ) averaged per phased loci from dataset 1. We also estimated the nucleotide divergence between pairs of samples from different sites using the average number of nucleotide substitutions per site (XYD) and the net divergence, corrected for the variation within analyzed samples (AD).Genetic diversity indexes were obtained with the software DnaSP v. 6.12.03 . We calcE. erythrinus, as measured by their impact on the model’s covariance. Therefore, when a given K value did not contribute anymore to the result, the previous K was selected [We assessed population structure with a principal coordinate analysis (PCoA) to visualize genetic diversity distribution in each karyomorph. Then, we implemented the fastStructure Bayesian approach . This meselected .E. erythrinus populations, i.e., many acrocentric and few bi-armed chromosomes. The karyotype presented 2n = 54 (6m + 2st + 46a), without differentiated sex chromosomes. In addition, up to three supernumerary B chromosomes were observed, which usually appeared like double-minute chromosomes and stained less than the standard chromosomes  and the subtelocentric pair 4, in addition to one chromosome pair with centromeric sites (pair 14). In turn, the only subtelocentric pair was observed with 5S rDNA sites on the telomeric region of the short arms. This same chromosome pair also bears 18S rDNA sites on its long arms, thus evidencing an unusual syntenic condition concerning these two classes of ribosomal DNAs .The set of our CGH experiments aimed to compare genomes among the different studied populations. The intra-karyomorph comparative hybridization of gDNA probes from A1 and A2 individuals produced essentially many overlapping signals, which highlights their genomic similarities  and suggests significant sequence homology, with some of them probably related to major rDNA sites. Some population-specific signals observed indicate some over-represented signals, probably linked to a higher copy number of repetitive sequences in some chromosomal regions a–d. On tSequencing generated around 2 million reads for each sample. After trimming and filtering steps, we obtained 14,467 phased loci with a minimum coverage of six present in all individuals, which we refer hereafter as dataset 1. Then, we created dataset 2 by maintaining only one biallelic SNP per sequence locus to avoid tightly linked SNPs, which resulted in 8597 polymorphic SNPs. Bayescan did not consider any locus as potentially under selection, therefore no marker was removed from subsequent analysis.The PCoA indicates a clear separation between karyomorphs A and D. Karyomorph D formed two minor groups that are closely related, and karyomorphs A1 and A2 formed one major cluster each, with some individuals scattered between these two clusters A.The non-geographic Bayesian approach fastStructure was congruent with the PcoA result and pointed out K = 2 as the value that maximizes likelihood and best explains the data. The analysis clustered A1 and A2 as a single population and karyomorph D as a second population B. The spWθ) were higher in the A1 locality, while the lowest values were present in D. All sampled groups presented negative Tajima’s D values, with the lowest in A1, followed by A2 and then D. Values of genetic differentiation, XYD and AD, were approximately ten times lower within karyomorph A when values between karyomorphs A and D were contrasted  and Watterson theta , conomosomes . The karomosomes .1 and X2 chromosomes and in the neo-Y chromosome [E. erythrinus, since both karyomorphs A1 and D reveal the same hybridization results for this probe. However, karyomorph A2 (herein described) presents six chromosomes mapped with the 18S rDNA probe . Differences in the distribution of the 18S rDNA among populations have already been described for fish of the same family, as in the case of Hoplias malabaricus . An invreviewed .E. erythrinus karyomorphs. Among fish, species with conserved karyotype macrostructures are also reported to have a variable pattern of genomic organization, as observed for Lebiasina [E. erythrinus that presents a differentiated sex chromosome (karyomorph D). This is in accordance with previous findings that the emergence of a new sex-linked locus or the translocation of an existing one is one of the fundamental steps in fish sex chromosome differentiation [The set of our CGH experiments aimed to compare genomes among the different studied populations. Such comparisons have been found to be very effective in identifying hidden biodiversity in fish (reviewed in ) and in ebiasina , for theebiasina  and for ebiasina . Differeebiasina , trahiraebiasina , pencilfebiasina  and charebiasina . In factntiation ,64. SuchE. erythrinus, therefore it is only possible to compare our results with those from other species. The mean value of nucleotide diversity (π) for E. eryhtrinus was 0.00077, while studies in other fish species gave higher values, as in Nassau Grouper (0.3566 with RAD-Seq data) [H. malabaricus with DArTSeq data (0.001172) [This is the first study that has investigated genetic diversity in eq data) . Convers.001172) . It is iXYD and AD values), it is evident that groups A1 and A2 are very similar, while karyomorph D is much more different from these groups, with values at least ten times higher. All groups showed negative Tajima’s D values, which measures the difference between the mean number of pairwise differences and the number of segregating sites. There are three main hypotheses for this result: (i) purifying selection is removing variation from these groups and maintaining their genetic diversity stably ; (ii) genetic drift may be influencing the genetic diversity of these groups, mainly because they are sedentary which may favor the occurrence of inbreeding [Moreover, our results indicate that karyomorphs A and D are already highly differentiated in both chromosomal and genomic features. When considering the two differentiation metrics implemented here  tE. erythrinus presents sedentary behavior and is not a migrant species. Taking all these facts into account, even though the two sampling localities are in the same geographic basin, they are somewhat isolated. When comparing karyomorphs A and D, we have a different scenario, since they are geographically distant from each other and are also different in terms of the presence of sex chromosomes, which could accelerate the differentiation of these populations and potentially lead to speciation.The current results and the divergence patterns observed are probably explained by two main factors. The first is that samples from the same karyomorph are expected to be more similar, although that is not a general rule. The second factor is the distance between these groups and, consequently, the probable time since the isolation event occurred. All karyomorph A groups are found in the Paraná River Basin, although they are in very distant regions. Additionally, Y-axis. This indicates that there is a higher diversity within and a low difference between the localities for karyomorph A. The Bayesian non-spatial population structure approach fastStructure  and D (multiple ♀X1X1X2X2/♂X1X2Y system) were collected in sintopy (the same location) and presented high genetic differentiation. According to values of XYD, the genetic differentiation between syntopic individuals from karyomorphs A and D was equivalent to the genetic differentiation of long-distance allopatric populations of karyomorph A. Other studies indicated that the emergence of neo-sex chromosomes could have contributed to the increase in genetic diversity , as demonstrated in sticklebacks [Rumex hastatulus [Among erythrinids, it has been recently highlighted in another study that the presence of a multiple sex chromosome system may be associated with the speciation process . Hoplias systems , couplin process . In H. mklebacks  and in tstatulus . In all statulus .E. erythrinus both from cytogenetic and genetic points of view. Even though the genetic diversity indexes were globally low, karyomorphs A and D are highly differentiated genetically. In addition, three main hypotheses for the differentiation found can be proposed. (1) Long-distance allopatry could have been the main agent of genetic differentiation between these karyomorphs. (2) The emergence of neo-sex chromosomes may have been the main factor that promoted this genetic differentiation. (3) Both long distance allopatry and the emergence of the neo-sex chromosomes on karyomorph D may have contributed to the fixation and accumulation of divergences. Hypothesis 3 is our main hypothesis. In summary, our data provided an additional layer of evidence for the status of the taxon E. erythrinus and corroborated previous studies, supporting the conclusion that it is probably a species complex with an unresolved taxonomy. Future studies using high-throughput sequencing focusing on sex determination in this complex will help to highlight the possible link of sex chromosomes with the speciation process in this group.Our results indicated the presence of high genetic differentiation among populations of the species complex


Posterior urethral valves (PUVs) and ureteropelvic junction obstruction (UPJO) are congenital obstructive uropathies that may impair kidney development.To identify genetic variants associated with kidney injury in patients with obstructive uropathy.We included 487 patients born in 1981 or later who underwent pyeloplasty or valve resection before 18 yr of age in the discovery phase, 102 PUV patients in a first replication phase, and 102 in a second replication phase.2, high blood pressure, antihypertensive medication use, proteinuria, and/or one kidney functioning at <45%. We used χ2 tests to calculate p values and odds ratios for >600 000 single-nucleotide polymorphisms (SNPs) in the discovery sample comparing patients with and without signs of kidney injury within 5 yr after surgery. We performed stratified analyses for PUV and UPJO and Kaplan-Meier and Cox regression analyses in the discovery and two replication samples for the associated SNPs, and RNA and protein expression analyses for the associated gene in fetal tissues.Signs of kidney injury were defined as dialysis, nephrectomy, kidney transplantation, estimated glomerular filtration rate (eGFR) <60 ml/min/1.73 mCDH12 gene was the most clear (p = 7.5 × 10–7). This SNP also seemed to be associated with time to kidney injury in the PUV discovery and replication samples. RNA expression analyses showed clear CDH12 expression in fetal kidneys, which was confirmed by protein immunolocalization.Despite the small and nonhomogeneous sample, we observed suggestive associations for six SNPs in three loci, of which rs6874819 in the CDH12 as a candidate gene for kidney injury in PUV.This study identified CDH12 gene increase the risk of kidney injury in patients with extra flaps of tissue in the urethra . This is the first report on this gene in this context. Our study provides interesting new information about the pathways involved and important leads for further research for this condition.We found that variants of the  CDH12 increase the risk of kidney injury in patients with posterior urethral valves. This is the first time that this gene has been reported on in this context. Our study provides interesting new information about pathways involved in obstructive uropathies and important leads for further research.We found that variants of  PUV affects only boys and occurs in one in 4000 live male births Knowledge about the molecular pathways involved in the pathophysiology of kidney injury in congenital obstructive uropathy has mainly been derived from neonatal rats and mice with induced complete unilateral ureteral obstruction (UUO). Kidney response to experimental UUO involves oxidative stress, monocyte infiltration, uncontrolled apoptosis of tubular cells, fibroblast accumulation, and increased deposition of extracellular matrix Most of the genetic studies described above focused on candidate genes, but a hypothesis-generating approach, such as a genome-wide association study (GWAS), may shed new light on pathways involved in the development of kidney injury in obstructive uropathy. In addition, genetic variants associated with kidney function decline could be used in prognostic modeling approaches to better predict long-term kidney function. As no such studies have been conducted so far, the aim of this study was to perform a GWAS for kidney injury in obstructive uropathy. We included patients with PUV and UPJO under the assumption that similar genetic variants are associated with kidney function decline in both obstructive uropathies.22.12.1.1AGORA  is a database and biobank in the Radboudumc  collecting questionnaire data, blood/saliva samples, and phenotypic information from patients with congenital malformations and their parents 2.1.2For replication purposes, we included patients treated in the Isala clinics, Zwolle or University Medical Center (UMC), Groningen, The Netherlands. Owing to registration issues, we were only able to identify patients treated in 2002 or later.2.1.32) was enrolled between 2010 and 2012 and followed until 2018 at 39 pediatric nephrology centers in eight European countries. We selected children with PUV as the underlying diagnosis for whom genotype data, longitudinal eGFR measurements, and information on treatment changes were available.The European replication sample was derived from the 4C study 2.22, high blood pressure, antihypertensive medication use, proteinuria, and/or one kidney functioning at <45%. In the European replication sample, the primary endpoint was kidney injury defined as a composite of 50% loss of eGFR, eGFR <10 ml/min/1.73 m², or kidney replacement therapy .The medical files for the Dutch patients were scrutinized for information about kidney function and for clinical descriptions. Patients were defined as having signs of kidney injury in case of dialysis, nephrectomy, kidney transplantation, eGFR <60 ml/min/1.73 m2.3The Dutch samples were genotyped by deCODE Genetics  using Infinium OmniExpress bead chips or Global Screening arrays . European replication samples were genotyped using Illumina Infinium 2.5M-8 microarrays. Quality control and imputation are described in the Supplementary material.2.4www.cog-genomics.org/plink/1.9/). Analyses were adjusted for the first four principal components to account for population stratification. We repeated the analyses with imputed genotypes for the area showing an association signal and visualized the results in LocusZoom (http://locuszoom.org/). For single-nucleotide polymorphisms (SNPs) passing the threshold of p = 1 × 10–5, we examined the associations separately for PUV and UPJO patients to verify the assumption of similar genetic variants associated with kidney function decline in these phenotypes, performed Kaplan-Meier and Cox regression analyses to include 130 patients with <5 yr of follow-up, and checked results in the replication samples. In additional analyses, we adjusted the Cox regression analysis for the most significant SNP in the PUV subgroup of the discovery sample for the possible prognostic factors from https://training.cochrane.org/online-learning/core-software-cochrane-reviews/revman/revman-5-download) using the inverse-variance method and a random-effects model.We excluded patients with <5 yr of follow-up from our discovery sample and compared patients with and without signs of kidney injury within 5 yr after surgery using the PLINK whole-genome data analysis toolset (2.5www.ebi.ac.uk/gxa/home) in February 2019 to see which tissues CDH12 is expressed in We used the Expression Atlas (www.proteinatlas.org/) in February 2019 We used the Protein Atlas  in February 2019 to see whether the SNPs that we identified are expression quantitative trait loci (eQTL) influencing expression of CDH12.Finally, we checked the GTEx portal  participated . We excluded 40 patients who did not have their first surgery (pyeloplasty or PUV resection) in the Radboudumc, four patients with end-stage kidney disease before surgery, and five patients without follow-up information. Of the remaining 535 samples, 12 failed genotyping and 36 failed sample quality control . Genotyping information for 487 patients was available for the discovery analyses .3.1.2For replication purposes, we identified 131 patients treated in the Isala clinics and 270 in UMC Groningen. Of these patients, 87 (66%) and 164 (61%), respectively, participated. We excluded three patients without follow-up information and 26 samples that failed quality control . We used genotyping information for 222 patients in the replication analyses.3.1.3The European replication sample was derived from a study including 704 children 3.2–5; p = 7.5 × 10–7). The other signals were from chromosomes 8  and 13 . Genotype frequencies were in Hardy-Weinberg equilibrium in controls , except for rs2957086 (p = 0.04). After imputation, rs6874819 remained the SNP with the strongest signal.We excluded 130 patients with <5 yr of follow-up and compared 141 patients with and 216 patients without signs of kidney injury within 5 yr after surgery. Six SNPs in three loci reached suggestive genome-wide significance (p < 1 × 10n = 235) and UPJO (n = 122) groups separately showed that the signals on chromosomes 5 and 13 were stronger in the PUV group, while the signal on chromosome 8 was stronger in the UPJO group (Supplementary Table 2).Subgroup analyses for the six SNPs in the PUV .In an additional analysis, we adjusted the Cox regression analyses for rs6874819 among the discovery PUV patients for the possible prognostic factors from 3.3GA 1.8, p = 0.11; HRGG 2.4, p = 0.40; GA 1.8, p = 0.11; HRGG 3.1, p = 0.04; Of the 222 Dutch replication samples, 78 were genotyped on the same platform as the discovery samples, while 144 were genotyped on the Global Screening array. Only two of the six suggestive SNPs (rs6874819 and rs2148707) were directly genotyped with the latter, so we used imputed genotypes for the others. We performed Kaplan-Meier and Cox regression analyses for 102 PUV and 120 UPJO patients, of whom 34 and 65, respectively, developed signs of kidney injury within 10 yr after surgery. Although not statistically significant, the associations of the SNPs on chromosome 5 were in the same direction as in the discovery sample for PUV patients . All studies in the Expression Atlas CDH12 shows the highest expression in human adult brain, whereas it is not expressed in adult kidney. The NIH Epigenomics Roadmap (http://www.roadmapepigenomics.org/) showed that CDH12 is expressed in human fetal kidneys CDH12 expression, whereas adult brain tissue does. Fetal kidneys showed expression levels comparable to those in adult brain. Transcriptomic analyses of 15 human embryonic tissues and organs 33–55 d after conception revealed that CDH12 is widely expressed in embryonic tissues, with highest levels in retinal pigmented epithelium, followed by the kidneys Visualization of the association results using imputed genotypes for the discovery sample in LocusZoom Immunohistochemical staining confirmed CDH12 expression in the fetal retina and kidney. At approximately 12 wk after conception, CDH12 was expressed in the ureteric bud, the precursor of the collecting duct, and in early condensates, where the ureteric bud induces mesenchymal cells to condense and start to form epithelia. In addition, CDH12 expression was localized to proximal tubules within the developing human kidney at 12 and 15 wk after conception .Fig. 3ImCDH12 in the adrenal gland. Several SNPs in the associated area resulted in higher expression of CDH12 . Two of these SNPs were directly genotyped in our samples. The CC genotypes resulted in statistically significantly higher expression of CDH12 in the adrenal gland according to the GTEx portal, and were associated with time to the development of signs of kidney injury in our data (Supplementary Table 3).The GTEx portal showed that the associated area was an eQTL for 4CHD12 as a gene potentially involved in the development of kidney injury in patients with obstructive uropathies. This result was seen among PUV patients, but not clearly among UPJO patients, possibly because of the different timing of PUV and UPJO development, or because both kidneys are exposed to high urinary pressure in PUV, whereas generally only one kidney is in UPJO.We identified for the first time CDH12 despite our small and nonhomogeneous sample suggests that it may be an important factor. This was confirmed by detection of the signal in the European PUV replication sample, which was a very different patient group. Although the primary results for rs6874819 were not statistically significant at a genome-wide level, the result for the allelic meta-analysis among PUV patients was. In addition, all results show the same direction of effect. The fact that our association signal is located in an eQTL and that CDH12 is expressed in fetal kidneys further enhances the reliability of our finding.The association signal on chromosome 5 among PUV patients was the most clear and the other suggested SNPs were not associated with kidney injury in the Dutch replication sample. Detection of the signal for In an additional analysis, we adjusted our analyses in the European replication sample for eGFR at study entry, which changed the effect of rs6874819 only slightly. Since eGFR at study entry correlates with GFR at birth, this finding suggests that the effect of rs6874819 on kidney injury we observed is not sufficiently explained by effects of CHD12 on fetal kidney development. Our hypothesis is that the damage arises in the period when affected kidneys are exposed to elevated pressure, which is from early fetal development up to the postnatal period preceding surgery. The damage may not be severe enough to display signs of kidney injury at that point in time, but as the patients grow and need greater filtration capacity, the affected kidneys may fall short and the previously acquired damage begins to show.CDH12 is a member of the neural cadherin (N-cadherin) gene family, which encode transmembrane calcium-dependent homophilic adhesion receptors that play an important role in cell recognition and sorting during development CDH12 has not been described in relation to kidney development before, but it is involved in the progression of several cancers MCP1 gene expression in these kidney tissues, which is in line with the suggestion by Diamond et al CDH12 is a target of MCPIP A drawback of our study is the relatively small sample size. We may have missed less strong but still important associations because our discovery analyses only had enough power (80%) to detect variants with an allele frequency of 30% and genotype relative risk of 2.7 or higher under a multiplicative disease model. In addition, use of a more homogeneous cohort of, for example, only prenatally detected PUV, or a more homogeneous endpoint, such as the eGFR decrease in the first yr of life, may have been better. Instead, we used any sign of kidney injury as the outcome to achieve larger numbers, but did not have enough patients with specific signs to perform separate analyses for subgroups. Nevertheless, when we performed additional analyses that excluded one kidney functioning at <45% from the definition of signs of kidney injury (the diagnosis that explains why more UPJO than PUV patients have signs of kidney injury), results for PUV patients in the discovery sample were very comparable to the initial results. Using a more homogeneous cohort would result in patients being more comparable for other factors that could influence the prognosis of PUV 5CDH12 variant, which facilitates monocyte infiltration and the development of progressive kidney fibrosis and exacerbates kidney injury. This hypothesis is in line with the importance of monocyte infiltration in the development of kidney injury in obstructive uropathy and provides leads for further research. The role of CDH12 in monocyte infiltration and the development of kidney injury in PUV, and the predictive value of rs6874819 for the prognosis of individual PUV patients are important future research topics, as is the generalizability of our findings to other forms of obstructive uropathy.We hypothesize that MCP1 pathway upregulation due to an increase in urinary tract pressure in PUV patients is augmented in carriers of the Author contributions: Loes F.M. van der Zanden had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.Study concept and design: van der Zanden, van Rooij, Renkema, Schreuder, Roeleveld, Feitz.Acquisition of data: van der Zanden, van Rooij, de Wall, Bongers, Renkema, Schreuder, Roeleveld, Feitz, Quaedackers, Nijman, Steffens, Schaefer, Kirchner, Behnisch, Bayazit, Caliskan, Obrycki, Montini, Duzova.Analysis and interpretation of data: van der Zanden, van Rooij, Renkema, Schreuder, Roeleveld, Feitz, Schaefer, Kirchner, Behnisch, Wuttke, Jennings, Hanley, Milmoe, Winyard.Drafting of the manuscript: van der Zanden.Critical revision of the manuscript for important intellectual content: van Rooij, Quaedackers, Nijman, Steffens, de Wall, Bongers, Schaefer, Kirchner, Behnisch, Bayazit, Caliskan, Obrycki, Montini, Duzova, Wuttke, Jennings, Hanley, Milmoe, Winyard, Renkema, Schreuder, Roeleveld, Feitz.Statistical analysis: van der Zanden, Behnisch, Hanley.Obtaining funding: van der Zanden.Administrative, technical, or material support: None.Supervision: Feitz, Roeleveld, Schreuder.Other: None.Financial disclosures: Loes F.M. van der Zanden certifies that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript , are the following: Rien J.M. Nijman has received lecture fees from AstraZeneca, Giovanni Montini has received consultancy fees from Merck, and Neil A. Hanley has received consultancy fees from APIS Assay Technologies, all outside the scope of the current manuscript. The remaining authors have nothing to disclose.Funding/Support and role of the sponsor: This research was supported by a Kolff junior postdoctoral grant from the 10.13039/501100002997Dutch Kidney Foundation (13OKJ36) and a ZonMW-VENI grant from The 10.13039/501100003246Netherlands Organisation for Scientific Research (91618036). The sponsors played a role in the design and conduct of the study.Acknowledgments: We would like to thank all those involved in data collection, all patients and parents for their cooperation in this study, and deCODE Genetics  for performing the microarray experiments. Part of the collaboration was supported by the European Reference Networks (ERNs) for rare urogenital diseases and complex condition (ERN eUROGEN) and for Rare Kidney Diseases (ERKNet). Fetal tissue was provided at the UCL GOS Institute of Child Health by the MRC-Wellcome Trust Human Developmental Biology Resource.Ethics considerations: This study was approved by the Regional Committee on Research Involving Human Subjects Arnhem-Nijmegen and by the board of directors of the Isala Clinics and University Medical Center, Groningen. The 4C study was approved by the central ethics committee of Heidelberg University Medical Faculty and by each local institutional review board. All participants and/or their parents provided written informed consent. The collection, storage, and use of human embryonic and fetal material were approved by the North West Research Ethics Committee or via the Human Developmental Biology Resource, under the codes of practice issued by the Human Tissue Authority and legislation of the UK Human Tissue Act 2008.