The ability of the adult mammalian heart to regenerate can save the cardiac muscle from a loss of function caused by injury. Cardiomyocyte regeneration is a key aspect of research for the treatment of cardiovascular diseases. The mouse heart shows temporary regeneration in the first week after birth; thus, the newborn mouse heart is an ideal model to study heart muscle regeneration. In this study, proteomic analysis was used to investigate the differences in protein expression in the hearts of neonatal mice at days 1 (P1 group), 4 (P4 group), and 7 (P7 group). Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed changes in several groups of proteins, including the protein kinase A (PKA) signaling pathway. Moreover, it was found that PKA inhibitors and agonists regulated cardiomyocyte replication in neonatal mouse hearts. These findings suggest that PKA may be a target for the regulation of the cardiomyocyte cell cycle.The online version contains supplementary material available at 10.1186/s12953-023-00219-4. Cardiomyocytes are terminally differentiated cells with very limited regenerative capacity. Several sources of injury can cause irreversible myocardial cell death, which results in the permanent loss of cardiac function and subsequently leads to cardiac diseases including myocardial infarction, malignant arrhythmias, heart failure, and sudden cardiac death . Heart iThe rate of cardiomyocyte renewal decreases with age and less than half of cardiomyocytes are renewed during the normal human lifespan . The regProtein kinase A (PKA) is a cAMP-dependent kinase. Once activated, it can phosphorylate various proteins and regulate cellular functions, including gene transcription, energy metabolism, cell cycle, and apoptosis, in almost all mammalian tissues . The PKAA previous study has confirmed that PKA overactivation leads to heart failure . In miceIn this study, we compared proteomic changes in newborn mouse hearts over time and found that the PKA signaling pathway is a target in regulating the cardiomyocyte cell cycle. To investigate whether the PKA signaling pathway regulated cardiomyocyte regeneration, we used a PKA inhibitor and agonist to treat primary cardiomyocytes. Our findings suggested that PKA activity was closely related to cardiomyocyte replication. Treatment with the PKA inhibitor could promote cardiomyocyte division, whereas treatment with the PKA agonist inhibited cardiomyocyte entry into the cell cycle.Neonatal C57BL/6 mice from specific families  were euthanized with pentobarbital and their hearts were excised for protein extraction.n = 4), P4 (n = 3), and P7 (n = 3) groups were homogenized in RIPA lysis buffer containing a protease inhibitor cocktail (Roche). The homogenates were centrifuged, and the Bradford assay was used to measure the protein concentration in the supernatant. To minimize biological variations, we used a pooled approach to extract protein. Equal weight ventricular tissues from the same day  were pooled together as described in previous studies ). P < 0.05 was considered significant. The significance of differences was determined using independent samples t-tests or one-way analysis of variance followed by Student-Newmann-Keuls multiple comparison tests.All statistical analyses were performed using SPSS software . Values are presented as mean ± standard error of the mean  were significantly higher in the P7 than in the P1 group  during PKA activation is an important step for its biological function . To stud+ from 13.03% to 7.13%, p = 0.0023; 7.78%, p = 0.0102)   Fig. A and C, Heart disease is the leading cause of death worldwide. Heart muscle cells in the adult heart lack regenerative capacity and, thus, damaged necrotic heart muscle cells are replaced by fibrous tissue, leading to decreased heart function . The undPrevious studies show that adult cardiomyocytes are generally highly differentiated, and damage to the hearts of mice older than a week is difficult to repair . In thisThe recent development of comparative proteomics technology has provided a technical platform to study the molecular mechanisms underlying myocardial regeneration, allowing the rapid accumulation of data on myocardial regeneration-related and signaling pathway–related factors . This hePrevious studies have shown that protein kinases regulate various cellular functions and as potential therapeutic targets for the management of cardiovascular diseases . As an iIn this study, we conducted a quantitative proteomic analysis of neonatal mouse hearts at day 1, day 4, and day 7. Comparison of the P4 group with the P1 group, a total of 3785 proteins were quantified of which 222 proteins were upregulated and 155 proteins were downregulated. Comparison of the P1 and P7 groups led to quantification of 3788 proteins, of which 258 were upregulated and 215 were downregulated in the P7 group compared with those in the P1 group. Bioinformatic analysis showed that proteins related to the cell cycle, DNA packaging, DNA repair, and ribosome biogenesis were significantly downregulated in correspondence with the development of the neonatal mouse heart, while proteins involved in cellular respiration, lipid metabolic process, ATP metabolic process, mitochondrion organization, cellular calcium ion homeostasis, phagocytosis, and the inflammatory response are upregulated from day 1 to day 7. KEGG pathway analysis showed changes in many protein groups, including those associated with the PKA pathway. Further study suggested that PKA activity was closely related to cardiomyocyte replication. Treatment with the PKA inhibitor could promote cardiomyocyte division, whereas treatment with the PKA agonist inhibited cardiomyocyte entry into the cell cycle. These results indicated that PKA may play a critical role in neonatal myocardial regeneration. These findings provide a new experimental foundation for pathological cardiac regeneration, suggesting novel treatment strategies.Additional file 1:Figure S1. Statistical enrichment test” results from the PANTHER website. Changes in protein enrichment between the P1 and P4 groups determined by the “statistical enrichment test.Additional file 2:Figure S2. Statistical enrichment test” results from the PANTHER website. Changes in protein enrichment between the P1 and P7 groups determined by the “statistical enrichment test. Additional file 3.


P < 0.001). In wet season, risk of vitamin (OH)D insufficiency was three times higher compared to dry season . The odds of vitamin (OH)D insufficiency was 11 times higher in fresh water areas compared to high saline areas . The girls had higher risk of iron deficiency in wet season. Despite the environment being enriched with micronutrient-contained aquatic food, adolescent girls in coastal areas experience different micronutrient deficiencies. The high prevalence of vitamin (OH)D insufficiency in freshwater locations and seasonal iron deficiency in high saline areas needs attention.Inadequate intake of both macro and micronutrients is the major determinant of micronutrient deficiencies in adolescent girls. This study assessed multiple micronutrient status including vitamin D, iron, vitamin A, and urinary iodine concentration among adolescent girls through two seasonal cross-sectional surveys conducted during dry and wet seasons. Mixed-effects linear and logistic regression analysis were conducted to assess associations between micronutrient status, salinity and seasonality. The mean age of the girls was 14 years. Vitamin (OH)D insufficiency was significantly higher in freshwater areas in wet season compared to dry season (wet season: 58% and dry season: 30%,  After early childhood, adolescence is the most critical period for growth and changes in body composition, physiology, and the endocrine system2. The majority of rural Bangladeshi women have their first child before reaching the age of 18 years, which frequently has a detrimental effect on both maternal and child health3. According to the Bangladesh Demographic Health Survey (BDHS) 2017–18, 24% of ever-married women aged 15 to 19 years old were chronically energy deficient [body mass index (BMI) < 18.5] and 9% were moderate to severely thin (BMI < 17.0)4. Hence, to break the vicious cycle of intergenerational malnutrition, adolescence is the last opportunity to catch up on any growth faltering experienced during childhood and support the growth spurt alongside skeletal development observed during this period2. Micronutrient deficiency is a major public health concern worldwide, with adolescents being one of the most susceptible groups5, primarily due to poor micronutrient content and bioavailability in diets, as well as poor hygiene and infections7. A household survey in Bangladesh showed that the median intakes of key micronutrients by pregnant adolescent girls were below the estimated average requirements (EARs)8. The requirement of micronutrients increase among adolescent girls during periods of peak physical growth when the body needs an increased input of dietary iron, calcium, zinc, iodine, vitamin A, and vitamin D9.Adolescence is a decisive life stage for achieving optimal health and nutrition11. Adolescent girls are particularly at risk in Bangladesh because 40% of the school children had mean UIC below 100 µg/l12. Vitamin D is important for calcium homeostasis to maintain skeletal integrity. A study in Bangladesh found an aging-related rise in both the prevalence of vitamin D insufficiency (S-25OHD 25–75 nmol/L) and the deficiency (S-25OHD < 25 nmol/L) among adolescents, with nearly all adolescents aged 12–16 years being either deficient (46%) or insufficient (52%)13. Both iron deficiency (ID) and iron deficiency anaemia (IDA) are global health problems. However, the adolescent girls14 are more likely to suffer from growth retardation15. According to a study, 32% of the adolescent girls living in peri-urban areas had iron deficiency anaemia16. Another essential micronutrient is vitamin A, which has a variety of physiological functions. Vitamin A deficiency (VAD) results in night blindness, severe anaemia, wasting, reproductive infirmity, infectious morbidity, and increased risk of mortality17. In Bangladesh, one-third of the non-pregnant and non-lactating populations are experiencing mild grade of vitamin A deficiency18. Hence, consumption of micronutrient-rich foods should suffice all micronutrient requirements.Iodine deficiency among adolescent girls can lead to goiter, mental and physical growth retardation which later on may extend upon their fetuses and newborns via intergenerational effect23. In Bangladesh, fish is consumed frequently, often daily or several times per week in peak seasons of high availability, although the portions consumed can be too small to provide a sufficient amount of key micronutrients24. The southwest coastal region of Bangladesh is considered one of the most diversified hubs of aquatic resources from fisheries and aquaculture25. In addition, the dynamics of the ecosystems in these coastal zones are complex, ranging from saline to freshwater aquatic environments, with seasonal fluctuations in the intrusion of oceanic water meeting riverine freshwater27. The availability of aquatic foods is highly diverse across the ecological zones, and it fluctuates between dry and wet seasons. Yet, there is no information available on how the delicate eco-system affects the seasonal nutrition and health status of the vulnerable people. We postulated that the overall micronutrient status of these girls may be impacted by salinity, seasonality, and consumption of various sea foods.Fish is significantly a good source of dietary protein and micronutrients in low- and middle-income countriesWe hypothesized that salinity, seasonality and intake of different sea food may have an impact on the overall the micronutrient status of these girls. Therefore, we undertook the study to assess the micronutrient status including urinary iodine concentration, total vitamin (OH)D, serum ferritin, and serum retinol. We also assessed food intake in adolescent girls living in aquatic farming communities across a gradient from coastal to inland environments, representing different ecosystems.28. The study population was adolescent girls aged 12–16 years old. Based on the dry season surface water salinity of adjacent rivers and canals, seafood production of the southwest coastal floodplain is divided into four major agro-ecological areas. Agro-ecologies that characterize the prevailing salinity gradient of the region were used to identify household clusters engaged in farmed seafood value chains; they included high-, medium-, and low-salinity, freshwater, and an urban location with a concentration of shrimp processing facilities29. The study focused on the two most important nodes of the seafood value chain in Bangladesh: the farming community and the processing zone. They are: (1) high saline area (HS); salinity is > 10 ppt (2) medium saline area (MS), salinity ranging > 5 to 10 ppt (3) low saline area (LS) typically the salinity remains 0.5–5 ppt (4) freshwater area (FW); salinity < 0.5 ppt28. Data collection covered a seafood processing plant (PP), a location where companies received cultured seafood products from the fish farms for processing and packaging for export markets. Sampling was stratified by the five settings , with 60 adolescent girls recruited in each site. Additionally, the sample was stratified for religion aiming for equal numbers of Hindus and Muslims in each area. Although at the national level, Hindu communities represent less than 10% of the population, the proportion of Hindus in the study areas was high. Sub-districts from each aqua-agroecology were purposively selected and local demographics were assessed after engagement with sub-district (Upazila) level officials. Key informant (KI) interviews led to identification of unmarried adolescent girls at household levels of selected communities. Communities with two adjacent Para , one Muslim-dominated and the other Hindu-dominated, were selected for each zone, considering the size of the community to ensure an adequate sample size. The PP area was predominantly Muslim (> 98%) and this community was therefore not stratified by religion. Households with at least one unmarried adolescent girl were identified and invited to participate in this study. The same girls were surveyed repeatedly, during the wet and dry seasons, for a total sample size of 300 adolescent girls in each season. In total, 298 teenage girls took part in the first round of data collection. During the second survey, however, the field team was unable to reach every girl who participated in the first survey. A total of 270 girls were surveyed, with a dropout rate of 9% due to marriage, absences during data collection and out migration Fig. Two stratified cross-sectional surveys were carried out from August—September 2017 (dry season) and April—May 2018 (wet season). The wet season is associated with peak shrimp production, and the dry season with peak fish and prawn productionThis study was conducted according to the guidelines laid down in the Declaration of Helsinki. Here, all of the study procedures involving the adolescent girls were ethically approved by the Ethical Review Committee of the icddr,b and NHS, Invasive or Clinical Research (NICR) Committee, University of Stirling, UK (PR-17037). Written informed consent was obtained from all participants and their parents or guardian before their participation in this study.A team of field research staff with relevant experience recruited by Noakhali Science and Technology University (NSTU) was responsible for the collection of field data on socioeconomic indicators, dietary intake, fish consumption, and anthropometric parameters using a structured and semi-structured questionnaire. The field supervisors and data collection teams were trained on the interview techniques, questionnaires, and nutritional status measurements. The data collection tools were pre-tested among the volunteer population, a total of 12 interviews were conducted. To maintain the standard of data collection, they adhered to anthropometry guidelines and a standard operating procedure (SOP). In addition to training, the data collectors completed a validation exercise. Blood and urine samples were collected by trained medical technologists from International Centre for Diarrhoeal Disease and Research .30. The amount of food consumed by the adolescent girls was calculated using pictures of the serving and the weight (in grams). The weight of the raw food was computed using the proper conversion factors12. According to accepted methods31, all food items were divided into ten major food groups. They are: (i) cereals , (ii) vegetables , (iii) pulses , (iv) eggs, (v) flesh food , (vii) milk (milk and milk products), (viii) beverages, (ix) fruits rich in vitamin A, and (x) other foods. The respondents were asked to recall which kinds of fish they had eaten regularly in the previous five days, three months, and one year. Fish intake  was also ranked at the household level by the types of fishes most frequently eaten. A wealth index was created using principal component analysis (PCA) of household assets information. A weight was attached to each item from the first principal component. The households were classified into SES quintiles based on the wealth index, from quintile 1 (poorest) to 5 (richest)32.Socio-demographic information included the level of education, occupation of the household head, number of family members, ownership of the house, number of dwelling rooms, household construction materials, toilet facilities, sources of drinking water, land ownership, monthly household expenditure, and household assets was collected as indicators of socioeconomic status (SES). The dietary diversity of adolescent girls was assessed using a 24-h dietary recall approach, and the types of fishes consumed were assessed using a 7-day semi-quantitative Food Frequency Questionnaire (FFQ), which had been previously designed and validatedThe adolescent girls were weighed using electronic scales  with a precision of 100 g. Height was measured using locally made standardized wooden length/height boards with a precision of 0.1 cm (1 mm). In both survey points, blood and urine samples were collected in a health center, developed as temporary sample collection centres on the same day. 3.5 ml of venous blood were collected in a Venoject tube to obtain 1200 µl serum. After the blood collection from all participants, the blood tubes were placed in a cool box and allowed to clot. At the end of each day, the whole blood was centrifuged and the serum was aliquoted into at least three cryovials by pipetting using a disposable pipette. A barcoded label was provided for each of the study participant’s questionnaire form and each of the aliquoted cryovials. The serum was stored in a freezer (-20ºC or colder) as soon as possible. During the transportation of the serum to the Nutritional Biochemistry Laboratory at icddr,b, the cold chain was maintained. Samples were stored in a -70 °C freezer and analyzed in the Nutritional Biochemistry Laboratory to estimate blood parameters. The participants provided urine samples in single-use plastic cups at the survey spot. The samples were transferred to wide-mouthed screw-capped plastic bottles that had been previously washed with de-ionized water and dried.33 at the icddr,b. A mean and/or median urinary iodine concentration (UIC) (µg/l) can be used to report the iodine status35. Urinary iodine concentration (UIC) was defined as a concentration level < 100 µgm/L34. Total vitamin (OH)D level was measured by electrochemiluminescence binding assay using a Roche Cobas e601 automated immune analyzer36. The assay employs a polyclonal antibody directed against 25-OH vitamin D37. Vitamin (OH)D insufficiency was defined as a serum level < 50 nmol/L and < 30 nmol/L for deficiency38. Serum ferritin, C-reactive protein (CRP) and alpha-1-acid glycoprotein (AGP) were analyzed by a sandwich ELISA technique39. Iron deficiency (ID) was defined as a median serum level < 15 ug/L, after adjusting for inflammatory markers CRP and AGP40. Serum retinol was determined using high-performance liquid chromatography (HPLC)41. The threshold for vitamin A deficiency (VAD) was a normal status: serum retinol ≥ 1·05 µmol/l (30 ug/dl), mild deficiency: serum retinol ≥ 0·7– < 1·05 µmol/l (≥ 20- < 30 ug/dl)42.Urinary iodine was determined by a colorimetric methodThe quality control team of investigators monitored the performance of the field personnel and supervisors through regular observations and regular checking of the data for completeness. The field supervisors independently repeated the data collection on socio-demographic information and anthropometry in a random selection of 5% of the study participants. Identical forms, equipment, definitions, and methods were used throughout the study period.2 P (1-P)/d2 (precision) × design effect. Here, Zα = 1.96 considering 95% confidence interval, P = 0.27 , d = 0.05 (5% level of significance) and design effect = 1.2. The total sample size came to 295 adolescent girls per survey. For ease of sample distribution across different study areas, a total of 300 adolescent girls was included in the study.The following formula for proportion in a cross-sectional survey was used to calculate the sample size: n = Zαχ2 test was used to compare the proportions of background and key outcome variables. We used a multivariable mixed-effect linear regression model with robust standard error to assess the association between the aquatic ecological zones and seasonality with the continuous measures for the status of the four micronutrients defined by the biochemical indicators as continuous variables . In addition, we used mixed-effects logistic regression to investigate factors associated with micronutrient deficiency defined by cut-off values for each of the micronutrients assessed . We specified a random effect at the individual level to account for within-individual correlations as data on the outcomes were collected on two time points. The “melogit” command in Stata was used to enable the inclusion of categorical covariates in the multivariable models. The regression was conducted using the stepwise backward method to determine the factors significantly associated with the occurrence of micronutrient deficiency. Each analysis (primary and secondary) was adjusted for age, dietary diversity quintiles, BMI, and wealth index. The data were reported by the regression coefficient and adjusted odds ratio (AOR) and 95% confidence intervals. Initial regression analyses included the interacting predictors and the independent variables that were supposed to interact as an interaction term. Model assumptions for each analysis were checked using residual and normal probability graphs. Non-normally distributed variables were logarithmically transformed before analysis. Data for urinary iodine, total vitamin (OH)D, ferritin and retinol were log-transformed prior to analysis.Data analysis was performed using STATA 13.0 SE . Pearson’s Table P < 0.005). Hence, compared to the dry season, the wet season tends to have a somewhat higher prevalence of vitamin (OH)D insufficiency. According to the vitamin (OH)D deficiency criteria [vitamin (OH)D < 30 nmol/L], 10% of the girls in the FW area had deficiencies during the wet season, but only 2% of the girls from the FW and PP areas had deficiencies during the dry season. Similar to vitamin D insufficiency, a rising tendency of ID was seen in the wet season as opposed to the dry season. Thirteen percent of the girls had ID in dry season and overall, one-fourth of girls from the HS areas were iron deficient during wet season. In the dry season, VAD (serum retinol < 30 ug/dL) affected 9% of girls in HS, MS, LS, and PP areas, but the prevalence of VAD was reduced in HS MS and LS areas in wet season. Nevertheless, in the FW area, it remained unchanged . Again, in these two regions, considerably more girls consumed prawn during the wet season compared to the dry season   among adolescent girls was higher during the wet season, although the difference was not statistically significant. The diversity of the diet varied significantly with the season; diets were more likely to contain food from more than five food categories during the wet season than the dry season . Girls from the MS and LS areas had significantly lower UIC than those from HS areas . On the other hand, girls who lived in the FW and PP zones had considerably higher levels of UIC. The total serum vitamin (OH)D level was lower during the wet season compared to the dry season . Vitamin (OH)D level was significantly higher among the girls living in MS area and lower in LS, FW and PP areas compared to girls from HS areas. When compared to the dry season, the serum ferritin was significantly lower in the wet season . Girls from PP areas were shown to have better ferritin status than those from HS areas. Even though there was a trend toward increased serum level concentration of vitamin (OH)D and ferritin during the dry season, mean serum retinol levels were noticeably higher than during the wet season. When compared to HS locations, the serum retinol level was significantly higher in MS areas.Table P = 0.003) areas. We found a significant seasonal effect on the likelihood of vitamin (OH)D insufficiency among the adolescent girls. The odds of vitamin (OH)D insufficiency was 3 times higher , P < 0.001) in the wet season compared to the dry season. Girls who resided in FW regions had a five-fold increased risk of vitamin (OH)D deficiency compared to girls who lived in the HS areas . The girls living in the FW and PP areas also had a higher risk of vitamin (OH)D insufficiency compared to the girls from HS area . The seasonal effect was also observed in the serum ferritin concentration of the girls; compared to the dry season, the risk of iron deficiency was around 4 times higher in the wet season . However, salinity level was not associated with risk ID. In comparison to the dry season, girls had a lower risk of vitamin A deficiency during the wet season .Table The study assessed micronutrient status and risks of micronutrient deficiencies among adolescent females residing in the coastal regions of Bangladesh. We found multiple micronutrient insufficiencies and deficiencies, which varied over a gradient from coastal to inland regions and with the seasons. One fourth of girls had ID in the wet season and 10% of girls had vitamin A deficiency in the dry season. In particular, the high prevalence of vitamin (OH)D insufficiency among adolescent girls living in processing plant areas during the wet season is of concern.13. In our study, we observed a higher prevalence of girls having vitamin (OH)D insufficiency especially in the freshwater zone across both seasons. More than half of the girls in the FW and PP areas had vitamin (OH)D insufficiency in the wet season. A study conducted in Iran reported women had significantly lower serum vitamin (OH)D concentrations than men in both summer and autumn (P = 0.021 and 0.016 respectively)44. Seasonal variability of vitamin D concentrations is also observed among the Polish children45. African-American girls had higher mean vitamin (OH)D levels in the spring/summer months than in fall/winter months46. Therefore, culture, religion, variation in sun exposure, clothing choice, skin pigmentation and age may be important contributors to total vitamin (OH)D level47. We observed a trend towards an increase in vitamin (OH)D insufficiency as the salinity of the area reduced. One plausible explanation could be the distinct dietary habits of the girls in the HS and MS areas, who had a higher intake of tilapia and substantially obtained the high levels of Vitamin D3. A study showed that farmed tilapia is a valuable source of vitamin D3; a study of the Polish population revealed lean tilapia has a high vitamin D content (38.0 ± 7.7 mg/100 g)48.In Bangladesh, vitamin (OH)D deficiency is a public health concern12. Half of the cases of anemia are accountable to iron deficiency, but the proportion varies among population groups and in different areas, according to the local conditions51. However, the prevalence of ID and IDA in the Bangladeshi population appeared to be substantially lower than the widely held assumption, perhaps linked to the bioavailable iron found in groundwater12. Remarkably, the iron status of the adolescent girls in this study differed with the season, with the highest prevalence of ID observed in the wet season in the three saline regions. A Brazilian study showed the mean serum ferritin concentrations of riverine populations were higher in the dry season than in the rainy season52. One reason contributing to the increasing levels of ID during the wet season may be the mixing of surface water and groundwater during the rainy season, which lowers the overall iron content of the mixed water53.In Bangladesh, the school-age children aged 6 to 11 and 12 to 14 years had an iron deficit of 3.9% and 9.5%, respectively54, and high consumption of fish by adolescent girls might have contributed to the adequate vitamin A status of these girls55. According to a study by Sabuktagin et al., eating animal source foods was associated with higher retinol levels in school children18.Twenty-one percent of the Bangladeshi school-going children are sub-clinically vitamin A deficient. In our study we found a low prevalence of VAD with seasonal fluctuation, girls living in the LS area had higher mean serum retinol levels (9%) compared to those in the HS area. Severe grade of vitamin A deficiency (serum retinol < 0·35 µmol/l) was not observed. Mandatory fortification of edible oil56. We found that none of the adolescent girls had urinary iodine concentration < 100 µg/L as the median level of urinary iodine concentration was higher than the population median level (> 100 µg/L) which indicates the girls are getting an adequate amount of iodine through salt, seafood and environment. The local diet and seafood may contain higher iodine concentrations in the HS areas possibly combined with a frequent presence of fish and seafood in the diet57. A study in China showed that the mean urinary iodine concentrations were significantly higher among children in coastal areas compared with children in inland areas in all age groups (P < 0.05)57. Another study reported a significant positive correlation between the iodine concentrations of salt samples and the urinary iodine concentrations of Bangladeshi adolescent girls and pregnant women43.Iodine Deficiency Disorder (IDD) is a significant global public health issue that affects all age groups, adolescent girls living in iodine-deficient areas had poorer school performance, lower IQs, learning disabilities and were more exposed to the consequences of IDD for their offspring59. Frequent consumption of tilapia may have played an important role in maintaining the normal concentrations of vitamin D3 among the adolescent girls.In terms of fish intake, almost 70% of the adolescent girls had consumed some fish in the last 24 h across all regions in both seasons. The seven-day food frequency data confirmed that tilapia was more commonly consumed in the HS and MS areas than in the LS, FW and PP areas; this finding can be explained by the abundance of tilapia in saline regions and its recent increase in commercial productionDespite the huge potential of fisheries and aquaculture to improve the nutrition situation in Bangladesh, the number of researches conducted is very low regarding the understanding of how aquatic environment and fish consumption are associated with nutritional status of the vulnerable groups, particularly the adolescent girls. Fish is a valuable contributor to the reference nutrient intakes for a variety of micronutrients. This study was such attempt to look into the factors contributing to different micronutrient deficiencies in adolescent females in Bangladesh's west coastal areas. For aquaculture ecoregions like Bangladesh, this research is crucial, thus we measured the micronutrient status, food intake, fish intake, and nutritional status of the adolescent, through two seasonal surveys. The study improved our understanding to establish a linkage among micronutrient status, aquaculture-ecological zone and seasonality.60. In addition to a distinct salinity zone effect, this study found seasonal variations in the prevalence of vitamin D insufficiency, vitamin A deficiency, and iron deficiency among adolescent girls living in coastal areas.One of the major limitations of this study is that we were only able to measure dietary diversity rather than adequacy. Hence, no conclusions can be drawn about to what extent dietary intake of adolescent girls influenced micronutrient status. Not measuring the hemoglobin level to estimate anemia prevalence among the girls is another limitation of the study. The influences of seasonality on food security, dietary diversity and nutritional status are widely accepted. A study in rural Bangladesh showed seasonality was a key determinant of dietary diversityAdolescent girls living across aquatic ecological zones in southern coastal areas of Bangladesh are seasonally at risk of multiple micronutrient insufficiencies and deficiencies. Attention should be paid to the high rates of vitamin (OH)D insufficiency, particularly in freshwater areas, and seasonal iron deficiency in the high saline zone. The results can support the development of targeted policies to improve the micronutrient status of this vulnerable group.Supplementary Information.


Then, genome edited variant alleles will be induced as long as Cas9 protein is active, and unmodified wildtype target loci are available. The corollary is that CRISPR-modified alleles that arise after the first zygotic cell division potentially could be distributed asymmetrically to the cell lineages that are specified early during morula and blastocyst development. This has practical implications for the investigation of F0 generation individuals, as cells in embryonic and extraembryonic tissues, such as the visceral yolk sac, might end up inheriting different genotypes. We here investigated the hypothetically possible scenarios by genotyping individual F0 CRISPants and their associated visceral yolk sacs in parallel. In all cases, we found that embryonic genotype was accurately reflected by yolk sac genotyping, with the two tissues indicating genetic congruence, even when the conceptus was a mosaic of cells with distinct allele configurations. Nevertheless, low abundance of a variant allele may represent a private mutation occurring only in the yolk sac, and in those rare cases, additional genotyping to determine the mutational status of the embryo proper is warranted.CRISPR-mediated genome editing  In its simplest instantiation, the CRISPR approach produces DNA breaks at a designated cut site theta, that the host cell then repairs through homology-directed repair (HDR) based on a template, or through non-homologous end-joining (NHEJ)2. In addition to its ease of use, a particular promise of the CRISPR technology lies in the simultaneous targeting of multiple gene loci5, to create genetic conditions that allow investigation of gene-gene-interactions without extensive and sometimes complicated breeding strategies6.The ability to target genome editing events to specific sites and the relative speed of application has brought CRISPR technology to the forefront for genetic manipulation in a wide variety of species7, or undesired inaccuracies at the intended targeted locus9. In approaches designed to rely on the repair of CRISPR/Cas9-induced DNA double strand breaks by non-homologous end-joining (NHEJ), small deletions of up to 10 base pairs can be expected at the target cut site theta10. However, we previously discovered that a single guide RNA targeted to the mouse T/brachyury gene, when co-injected with Cas9 protein into fertilized mouse oocytes, can generate numerous deletion variants with breakpoints even far away from the target site theta, and of varying length and direction11.However, widespread use in the past years has also revealed weaknesses of the CRISPR technology, such as the potential for creating off-target events11.In addition, we detected multiple variant alleles within the same animal, implying that the first Cas9-guideRNA-induced DNA break occurred only on one chromosome, and that additional mutations were induced during further cell division(s). Since any diploid cell can only contain two alleles of any given gene, any individual with 3 or more distinct alleles has to be a mosaic, composed of cells that contain distinct CRISPR induced mutant alleles12 that contribute to the visceral yolk sac after implantation13. Examination of F0 individuals becomes necessary when the targeted gene of interest is suspected or known to cause lethality at embryonic or fetal stages14; in those approaches, the associated visceral yolk sac is typically used for genotyping. Because the progenitors of visceral yolk sac endoderm are specified by the early blastocyst stage, it is theoretically possible that the yolk sac contains different variant alleles than the embryo proper. In a scenario with multiple consecutive CRISPR-induced mutation events, the yolk sac then may or may not inform appropriately about embryonic genotype of individual F0 CRISPants.Such a situation presents a particular complexity for the investigation of F0 generation individuals, particularly at embryonic or fetal stages, because specification of the inner cell mass -which gives rise to the embryo proper- occurs as early as the 16-cell morula stage, and by the 32-cell stage, the first primitive endoderm cells are detectable11. This guide RNA targets exon 3, which encodes a part of the DNA-binding domain of the T transcription factor. We showed that disruptions of this exon by CRISPR-induced deletions produce similar morphological anomalies as observed in conventional T mutant embryos11: a wide variety of defects in mesoderm derivatives, tails that are shorter, curled or absent, abnormal neural tube closure, kinked or wavy neural tubes15.In the present study, we conducted explicit tests for this possibility, by genotyping both embryo and yolk sac from the same conceptus at midgestation, using a validated guide RNA directed to the same target site in the T/brachyury locus, published previously11, but also contained distinct new alleles. Moreover, we failed to detect unique mutant alleles in the embryo that weren’t also present in the yolk sac. Taken together, our present results indicate that yolk sac genotyping appropriately reflects embryonic genotype in F0 mouse CRISPants.These phenotypes are also observed in our newly generated independent F0 CRISPants, which recapitulate some gene editing events identified in our previous study16 were quarantined for two weeks after arrival and fed a Purina Chow diet. These mice were used as zygote donors and super ovulated by intraperitoneal injection of 5 IU PMSG (ProSpec-Tany TechnoGene Ltd) one hour past the middle of the light cycle two days before breeding. One hour before matings to FVB/N males were set up, females were injected with 5 IU Human Chorionic Gonadotropin (ProSpec-Tany TechnoGene Ltd). If copulation plugs were detected the next morning, this was counted as day E0.5 of gestation.All mice  were housed in individually vented cages in a 7-hour dark/light cycle. Female mice of the FVB/N mouse strain17 and fed a Purina Breeder Chow diet (LabDiet 5015). They were used as surrogates at >than 6 weeks of age, after mating with a vasectomized CD-1 male to induce pseudopregnancy.Females of the outbred CD-1 mouse strain were used as foster damsAll animal husbandry was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institute of Health of the United States of America, covered by protocols approved by the Institutional Animal Care and Use Committee of the Pennington Biomedical Research Center.® Advanced KSOM Embryo Medium (EMD Millipore) at 37°C in an atmosphere containing 5% CO2 before and after microinjection. Microinjections were performed as previously described11 and consisted of 25 ng/μl TrueGuide™ sgRNA and 50 ng/μl TrueCut™ Cas9 Protein v2 (Invitrogen) diluted in EmbryoMax® Electroporation Buffer (EMD Millipore). The sgRNA targeting T/brachyury exon 3 was the same as in our earlier publication11. A maximum of 28 injected zygotes were transferred bilaterally into the infundibulum of the uterine horns of pseudopregnant CD-1 females. Surrogate females were euthanized at E10.5, and conceptuses were recovered for morphological analysis and genotyping.Fertilized oocytes at the single-cell stage were harvested from FVB/N females on E0.5 and cultured in EmbryoMaxConceptuses were isolated at E10.5 within their individual decidua, and placed in separate fresh dishes of OptiMEM (Invitrogen) for dissection. Embryos and their corresponding visceral yolk sacs were dissected apart and kept as individual samples for genotype analysis. Imaging of embryos was performed using a Leica S9i stereo microscope, and documented using V4.12.0 Leica Application Suite software (Leica Microsystems Co.). Samples were rinsed in 500μl sterile PBS and stored in 100μl PBS at −20°C until genomic DNA isolation.Stored samples were equilibrated to room temperature. To each sample 100μl of 2X lysis buffer  was added. Proteinase K digestion (10μl of a 10 mg/ml stock solution) was performed at 55°C overnight using a Thermomixer R (Eppendorf) set at 750 rpm. The next morning, 10μl 8M LiCl, 1μl Glycogen and 200μl Isopropanol was added to each sample, vortexed and incubated for 30min at −20°C. After centrifugation (Eppendorf Centrifuge 5417R) at 14000rpm for 30 minutes at 4°C supernatant was discarded and the pellet washed with 70% Ethanol. All Ethanol was removed after another centrifugation step and the pellet was air-dried for 15 minutes. DNA pellets were resuspended in 20μl LoTE buffer  and incubated for 15 minutes at 55°C in a Thermomixer. DNA concentrations were determined using the Qubit dsDNA high sensitivity kit on the Qubit fluorometer instrument (Invitrogen).PCR amplification was performed in 50μl reactions using a T100 Thermal Cycler (BioRad). The PCR primer pair was ordered from IDT (Integrated DNA Technologies). Reactions consisted of 2μl gDNA (25 ng/μl), 2μl forward primer , 2μl reverse primer , 19μl water and 25μl Long Amp Taq 2x master mix (New England BioLabs). Cycling conditions were 3 minutes at 95 °C, followed by 30 cycles of 15 seconds melting at 95 °C, 20 seconds annealing at 59 °C, 6 minutes and 30 seconds extension at 65 °C, with a final step of 10 minutes at 65 °C and a hold at 10 °C. The amplicons (6.58 kb) were purified with the QIAquick PCR column purification Kit (Quiagen) according to the manufacturer’s manual. Concentrations were determined using the NanoDrop One instrument (Thermo Scientific).18. Input DNA ends were prepared for barcode adapter attachment followed by a 17-cycle PCR amplification using barcoded primers and LongAmp Taq 2x master mix (New England BioLabs). Samples were pooled, DNA ends prepared for sequencing adapter ligation and loaded into the flow cell. All DNA purification steps were carried out with AMPure XP beads (Agencourt). The MinKNOW 21.11.8 version software was used for base-calling at high accuracy setting and created fastq files, which were merged for every sample before submitting the data to CRISPresso2 software for analysis.Libraries were generated using the PCR barcoding (96) genomic DNA Kit (SQK-LSK109) and sequenced on the MinION Portable Sequencer (Oxford Nanopore Technologies)19 was used to analyze obtained CRISPR edited sequences for the T/Brachyury gene, using default settings and Crispresso1 mode. Sequences of the sgRNA (ACTCTCACGATGTGAATCCG) as well as PCR amplicons were provided for CRISPResso analyses.The CRISPResso version 2.2.9 software11.We previously showed that editing of the T/brachyury gene by CRISPR methodology results in a wide spectrum of deletions that were only accurately detected by long-read single molecule DNA sequencing. Here, using the same methodology, we sought to determine, for new independently generated CRISPants, to what extent CRISPR-induced gene edits in the yolk sac can accurately inform about the embryonic genotype. To this end, fertilized oocytes of the FVB/N strain were microinjected with single guide RNA and Cas9 protein, and transferred into the oviducts of pseudo-pregnant recipient females of the CD-1 strain that served as surrogates. Having performed multiple injection sessions, we report here on a single such experiment, from which we recovered 10 successfully implanted conceptuses at day E10.5 (counted relative to the day of donor oocyte harvest). Yolk sacs were dissected away from the embryos proper, and the samples originating from the same conceptus were processed in parallel. Embryos were inspected for microscopically visible malformations, which were documented by digital imaging. Then, genomic DNA was prepared from all visceral yolk sac and embryonic samples, and subjected to PCR amplification and long-read single-molecule DNA sequencing of amplicons as described previouslyWe identified variant alleles in 4 yolk sac/embryo sample pairs, while the other conceptuses yielded only wildtype sequences, as shown in The relative proportions of allele modifications in sequences from each tissue are displayed in pie charts , Panel AIn sample pair 29N-F, the proportion of wildtype sequences is 56.5% and 57.2% in yolk sac and embryo, respectively. The unequal ratio of mutant to wildtype alleles again suggests that the individual is a mosaic, although an additional mutant allele is detected at low abundance in the yolk sac see . DespiteWe also here find multiple modified alleles in the same individual, specifically in sample pairs 29N-E and 29N-G1, which each contain two CRISPR-modified alleles and the wildtype allele. For sample pair 29N-E, yolk sac and embryo revealed 36.8% and 42.2% of sequences with the Δ11bp@-5 deletion, and 34.6% and 37.7% of sequences with the 1bp insertion at @1, in addition to 26.5% and 20.1% of wildtype sequences, respectively. Because heterozygosity for a mutant allele and wildtype would yield a higher representation of wildtype than is observed here, we have to conclude that the two mutant loci co-occur in the same cells, a condition termed “compound” homozygosity. Thus, this individual again is a mosaic, composed of cells that contain the two mutant alleles, and of wildtype cells. Based upon sequence representation, the proportion of mutant cells is approximately 75%, with only 25% wildtype cells, indicating that the mutations were induced at the two-cell stage, and that the mutant cell descendants subsequently gave rise to the greater share of embryonic cells. The embryo of this sample pair , Panel DIn sample pair 29N-G1, 59.1% of the yolk sac and 77.8% of the embryo sequences represent the largest deletion Δ1093bp@-2, while the 1bp insertion +1@1 appears only in 20.8% and 9.5% of yolk sac and embryo sequences, respectively. Intriguingly, these frequencies are closely mirrored in the proportions of 20.1% and 12.7% wildtype sequences in yolk sac and embryo, respectively, suggesting that the insertion co-occurs in heterozygosity with the wildtype allele in the same cells. Then, the deletion allele would be predicted to be in homozygous configuration, which would make up the vast majority of cells in the embryo, and still a large majority in the yolk sac. Consistent with the interpretation that mutant cells predominate in the embryo is its phenotype  Panel E:Taken together, our results reveal CRISPR-induced gene edits at the T/brachyury locus that recapitulate mutations from our prior discovery in an independent experiment, and also produce distinct new mutations. In each mutant conceptus, the allele frequencies reveal unique genetic configurations, contributing to different cellular compositions of the embryos, all of which were mosaics, with distinctive consequences for the observed morphological phenotypes. Importantly, the results provide unequivocal evidence that mutant alleles that occur in the embryo are also represented in the corresponding yolk sac.11. Having detected up to 7 distinct alleles, including wildtype, in the same individual, we reasoned that creation of these discrete events must have involved up to 7 cell divisions, during which the embryo could have grown to 128 cells (27=128), the size of the blastocyst prior to implantation. In vivo, this process can take up to 3 days, during which the separate germ layers are specified , but we here also identified 3 new distinct alleles . In contrast to our previous experience11, where all 39 out of 39 analyzed conceptuses were CRISPants with mutant alleles, we here detected modifications in only 4 out of 10 conceptuses. This could be due to a different batch of Cas9 enzyme, or the occurrence of deletions that would not be detected in our genotyping assay, e.g. if one of the binding sites for the amplification primers was deleted. This would be the case if deletions extended over 3.3 kb from theta in either direction. Large deletions have been reported in the literature27 for cultured cells and for whole organisms, as much many as over 0.5 Mbps were unintentionally deleted- 22 in a transgenic approach, similar to the one pursued in this study. Yet, the systematic application of long-read sequencing to CRISPR genotyping has been pursued only by a few research groups so far30, and the present study; conventional CRISPR genotyping overwhelmingly uses techniques that survey for edits only in a narrow envelope around theta34, and therefore miss events that create large deletions, inversions and other structural mutations. Nevertheless, the sample number of 4 CRISPants is sufficient to draw valid conclusions, according to the long-standing convention in the transgenic mouse field that three independent genetic events yielding the same outcome establish the observed outcome as caused by the genetic manipulation35, as opposed to some random manifestation, which would not be expected to occur consistently among multiple individuals.In light of several theoretically possible scenarios, this study therefore sought to determine whether yolk sac genotyping appropriately reflects embryonic CRISPR-induced genomic editing events. We detected CRISPR edits in 4 pairs of embryo and corresponding yolk sac. Interestingly, 3 of the 6 discrete CRISPR modifications are identical to those found in multiple individuals in our prior report11, in which at least 34 out of 39 individuals were mosaics, predicted from allele abundance in the yolk sac. Ascertainment of cellular composition and genetic configuration is particularly important in experimental approaches that have to investigate the F0 generation43, such as e.g. when permanent lines of mutant animals cannot be derived due to embryonic or perinatal lethality after mutation, or e.g. when multiple loci are targeted simultaneously that would segregate with breeding. Where live offspring result and can be propagated, the next generation individuals will contain only those alleles transmitted through the germline, and therefore all of their cells will have a uniform genotype35.Single-molecule sequencing in this study not only ascertained the particular CRISPR-mediated edits, but also yielded information on the relative representation of these edits among all recovered DNA sequences of the T/brachyury locus. The proportions of discrete alleles allow us to predict particular genetic configurations and cellular composition for each conceptus, all of which must have been mosaics. This is consistent with our earlier study11, based on yolk sac genotyping. The hypothetical possibility where the embryo contains alleles not found in the yolk sac (shown in 46- from a comparatively large number of progenitor cells, the scenario that they would all be wildtype is highly unlikely.With regard to our overarching question, namely to what extent yolk sac genotyping produces an appropriate reflection of embryonic genotype, our results provide unequivocal evidence: all discrete alleles detected by DNA sequencing in the embryo were also, in all cases, present in the yolk sac. This is consistent with the presence of mesodermal cells in the visceral yolk sac, which derive from the embryo and therefore would be expected to contain alleles that are also edited in the embryo  Panel B.shown in , Panel E11. In cases of low abundance of variant alleles then, subsequent genotyping of embryonic DNA would be warranted to obtain an accurate determination of embryonic CRISPR-induced edits and mutations, and for interpretation of phenotypes in mosaic F0 individuals6.On the other hand, the potential outcome where yolk sac might contain alleles not present in the embryo , Panel F15, and compared to the classical T/brachyury mutant48, our mosaic T-edited CRISPants display the same spectrum of developmental defects, including absence of somites or misshapen somites, abnormal development in the posterior region, open neural tube, and kinked neural tube. Thus, by targeting the T/brachyury gene, we were able to make evident the yolk sac-embryo correlations of genomic edits, and demonstrate their relevance in body structures whose development is dependent on mesoderm generated during gastrulation and on the subsequent proper migration of mesodermal derivatives50.Finally, we show that the CRISPR editing strategy produces morphological anomalies in the manipulated embryo, dependent on the proportion of cells with mutant alleles within the mosaics we recovered in this study. Compared to chimeras between T-mutant and wildtype cells generated by conventional methodsWe recovered CRISPR/Cas9-induced mutations at the T/brachyury gene in F0 CRISPant mouse embryos, and showed that their associated visceral yolk sacs contained mutant alleles that correctly reflect embryonic genotype. This is particularly relevant for diagnosis and interpretation of morphological anomalies that arise as a consequence of CRISPR modifications, when only the F0 generation is available due to subsequent peri-natal lethality caused by the induced mutations.Furthermore, the abundance of mutant alleles detected by single-strand long-range sequencing in this and our prior study indicated that all CRISPant embryos were mosaics of wildtype and mutant cells, in different configurations of hetero- and homozygosity. We discuss these results in light of cell specification processes in pre-implantation development, and highlight a particular scenario in which -in addition to the routine genotyping of yolk sac tissue- embryonic tissue should also be tested to achieve accurate genotype determination.51 on limitations of CRISPR/Cas9 genome editing in vivo.Our discovery of new edits that were not detected in previous studies, and the high prevalence of mosaicism observed in our CRISPants, add to the growing literature


Therefore, we have characterised the response of one susceptible and two resistant durum wheat accessions against leaf rust under different environments in greenhouse assays, simulating the predicted conditions of elevated temperature and [CO2] in the far future period of 2070–2099 for the wheat growing region of Cordoba, Spain. Interestingly, high temperature alone or in combination with high [CO2] did not alter the external appearance of the rust lesions. However, through macro and microscopic evaluation, we found some host physiological and molecular responses to infection that would quantitatively reduce not only pustule formation and subsequent infection cycles of this pathogen, but also the host photosynthetic area under these predicted weather conditions, mainly expressed in the susceptible accession. Moreover, our results suggest that durum wheat responses to infection are mainly driven by temperature, being considered the most hampering abiotic stress. In contrast, leaf rust infection was greatly reduced when these weather conditions were also conducted during the inoculation process, resembling the effects of possible heat waves not only in disease development, but also in fungal germination and penetration success. Considering this lack of knowledge in plant-pathogen interactions combined with abiotic stresses, the present study is, to the best of our knowledge, the first to include the effects of the expected diurnal variation of maximum temperature and continuous elevated [CO2] in the durum wheat-leaf rust pathosystem.Durum wheat cultivation in Mediterranean regions is threatened by abiotic factors, mainly related to the effects of climate change, and biotic factors such as the leaf rust disease. This situation requires an in-depth knowledge of how predicted elevated temperatures and [CO Therefore, the predicted expansion of the global population up to 9.6 billion people by 20502 would imply an increase of the global demand of wheat consumption. However, accomplishing this objective of production is a challenging task because current wheat yields are limited by diverse abiotic and biotic constraints.Wheat is considered one of the most important crops in the world, accounting for 771 million tons of production on 221 million hectares in 2021 and represents an essential source of calories and protein in the human diets4. Indeed, environmental conditions have suffered variations due to global climate change5, which could be considered an important constraint for plant growth and development through abiotic stresses such as high temperature and drought6, reducing wheat yield by 20–30% due to the impact of extreme events4. Concretely, the increase in carbon dioxide concentration [CO2] is expected to increase global temperature by up to 3.7 °C5, and, according to projections for Europe, this increase could be between 4.5 and 5.5 °C, depending on the CO2 emission scenario7. In addition, wheat is constantly exposed to biotic stresses, which are a major constraint to wheat production worldwide10. Amongst them, plant diseases cause more than 21% of wheat losses on average9, with fungal pathogens such as wheat rusts considered to be the most detrimental13.Abiotic stresses, such as drought or high temperatures, affect wheat cultivation depending on the region and environmental conditions, causing physiological and biochemical alterations that ultimately reduce wheat yields, even more than biotic stressesPuccinia triticina Erikss., is one of the most common and harmful rust of wheat15. Leaf rust infections reduce the photosynthetic activity of infected leaves, which usually results in yield losses  through decreasing number of kernels and lower kernel weights16. Currently, the most efficient, sustainable, and nonchemical approach to face leaf rust is through genetic breeding17. In fact, several genes of resistance to P. triticina have been isolated, identified and named as Lr genes (~ 80)18. Some genes are race-specific, acting in a gene-for-gene manner with the pathogen, and are associated with a qualitative hypersensitive response (HR), while others are non-race specific and develop a partial resistance response (PR), showing durable incomplete resistance, characterised by a slower development of the disease19. Thus, to discover potential sources of resistance, the identification of both macro and microscopic components of resistance is considered essential. In fact, although leaf rust evaluation has been generally achieved through visual assessment20, image analysis has emerged as a precise tool for quantitatively evaluating wheat rusts23. Additionally, histopathological methods allow the identification of microscopic components of resistance to wheat rusts such as generation of reactive oxygen species (ROSs), or the presence and development of a hypersensitive response (HR) amongst others25. In summary, both macro and microscopic evaluations are necessary not only in finding new sources of resistance, but also in the detailed evaluation of effects of abiotic factors in wheat-leaf rust interactions under future climate conditions.Leaf (brown) rust, caused by the biotrophic fungus 2] and water regimes17, which would modify plant development and resistance pathways, on one side, and pathogen virulence mechanisms and life cycle, on the other side26. Thus, disease risk simulation studies postulate as an essential tool to predict these climate impacts on wheat-pathogen interaction worldwide29, but they involve a certain degree of uncertainty30. This, combined with a lack of realistic field studies about the effects resulting from the combination of simultaneous abiotic and biotic stresses in wheat32, make disease predictions under future climate change a challenging task. In fact, these studies are far from finding a similar response about the effects of expected abiotic factors, such as high temperature and elevated [CO2], on wheat-pathogen interactions39, possibly due to diverse plant genotypes, pathogen lifestyles, and/or timing and intensity of simultaneous abiotic factors32. In this sense, elevated temperature modulates plant resistance against pathogens, increasing or decreasing it in terms of both basal and race-specific resistance40. In the case of wheat, it is known that some resistance genes against the most important rusts  are influenced by temperature, being effective only at warm environmental conditions43. As a consequence, the deployment of this kind of genes requires experiments with specific conditions of increasing temperature in wheat-rust interactions for their identification46, which would become complex in future breeding studies if weather conditions become warmer.Indeed, wheat-pathogen interactions are likely to be influenced by future climate change alterations in temperature,  in the far future period of 2070–2099 for the wheat growing region of Cordoba, Spain.The aim of this study was to identify macro and microscopic components of resistance against leaf rust in durum wheat accessions with different levels of resistance, using greenhouse experiments that simulated the predicted conditions of increased temperature and , similarly to the study of Porras et al.58 for evaluation of Septoria tritici blotch disease. To establish these weather and [CO2] conditions, the greenhouses were equipped with air conditioning and dehumidification systems, and CO2 supply circuits, all controlled by temperature, humidity, and CO2 sensors, with a fully automated CO2 injection process to maintain the CO2 target levels . The established weather conditions were designed to resemble a standard spring day, which is the expected growth period of P. triticina in the wheat growing area of Cordoba.Plants of the three selected durum wheat accessions were grown in greenhouses with full environmental control of temperature and  was set at around 420–450 ppm, the level currently observed outdoors. Moreover, in order to define the weather conditions for the far future period (2070–2099), the Representative Concentration Pathway RCP8.5 and an ensemble of five climate models  were taken into account, resulting in an average maximum and minimum temperatures around 30 °C and 15 °C, respectively, and an average [CO2] around 620–650 ppm.Since average temperatures may not always be an accurate predictor of the potential for an infection2] conditions, five sets of plants from three durum wheat accessions were exposed to three different environments, each in separate greenhouses, to assess P. triticina infection. Under baseline conditions (environment B), plants were exposed to a maximum temperature of 24 °C and [CO2] around 420–450 ppm. For the far future scenario, two possible environments were established: under increasing temperature (environment 1), plants were exposed to a maximum temperature of 30 °C and [CO2] around 420–450 ppm; and under increasing temperature and [CO2] (environment 2), plants were exposed to a maximum temperature of 30 °C and elevated [CO2] around 620–650 ppm.Having established the above weather and [CO2]. As high temperatures could affect critical phases of the P. triticina infection process, two of the four sets of plants under far future conditions were inoculated and incubated under baseline weather conditions, before returning to their respective far future conditions . The other two sets of plants were grown, inoculated, incubated and maintained for evaluation under their corresponding far future weather conditions .One set of plants was grown, incubated and maintained for evaluation under baseline weather conditions (set SB), and four sets of plants were grown under far future weather conditions: two sets at elevated temperature and two at both elevated temperature and  due to climate change, would lead to an uncertain scenario for durum wheat cultivation in the short and long term, especially in hotspots of climate change such as Mediterranean countries56. Although recent studies have evaluated the effects of elevated temperatures63 and elevated [CO2]38 in wheat-leaf rust interaction, not many have conducted diurnal fluctuating temperature cycles or even the combination of two abiotic factors. In this study, previous to the development of climate change studies, we first conducted an evaluation of leaf rust disease symptoms in a collection of 45 Spanish durum wheat breeding lines and commercial cultivars. Our results showed a general susceptible response (IT 7–9) amongst evaluated accessions, possibly due to the recent emergence of new virulent races in recent years not only in Spain55, but also in other Mediterranean countries such as France64. Particularly, the fact that the majority of breeding lines evaluated expressed a susceptible response against leaf rust is a quite concerning fact for the current Spanish breeding programs of durum wheat, which presented barely any sources of resistance against leaf rust65 in comparison with bread wheat66. However, some accessions, both breeding lines and commercial cultivars, expressed varied resistant responses, being valuable sources of resistance in future breeding programs66. Once all accessions were evaluated, we selected breeding line BL 38 (IT 1) as highly resistant, breeding line BL 28 (IT 4) as moderately resistant, and commercial cultivar Qualidou (IT 9) as susceptible for the development of climate change experiments.Durum wheat cultivation is currently threatened by abiotic and biotic stresses. In fact, the increased risk of wheat leaf rust, coupled with the effects of increased temperature and , but its effects vary from increasing39 to decreasing73 the incidence of different wheat diseases. In addition, few studies consider the effects of both elevated [CO2] and temperature together37, which would most closely resemble the effects of expected field climatic conditions in wheat-leaf rust interactions26. For that reason, plants of selected accessions were exposed to both elevated temperature and [CO2]  to assess their effects in the durum wheat-leaf rust interactions. To ensure infection, plants of this assay were inoculated and incubated during 24 h under baseline weather conditions (plant set S2).One of the most studied abiotic factors in plant diseases is elevated [CO2] for prolonged periods (days to weeks) reduces stomatal conductance and evapotranspiration, thereby increasing canopy temperature74. This situation forced S2 plants to develop a unique response during a subsequent leaf rust infection, which may reduce fungal penetration success through a partial stomatal closure76, confirmed by reduced IF values in Qualidou and BL 28 accessions in comparison with S1 plants. Despite this reduction, the IF parameter remained higher in the Qualidou accession (susceptible) compared to SB plants33. In addition, elevated [CO2] stimulates photosynthesis, especially in C3 plants as wheat6, increasing the production of sugar, starch and other carbohydrates77. On one hand, this additional carbohydrate accumulation in the leaf tissue could facilitate nutrient acquisition by the fungus76, as it was observed in the Qualidou accession with a reduction of the LP50 value under environment 2, in agreement with faster growth of other biotrophic fungus under elevated [CO2] conditions73. On the other hand, this carbohydrate production might act as an elicitor of defence responses such as enhancement of ROSs network and phytohormonal control75, as it was detected in Qualidou plants lower values of CS and EST−, coupled with higher values of EST + , and reduced values of Mean Pustule Size, Total Pustule Area, and Pustule Development Rate compared to SB plants.Exposure of plants to elevated [CO2]) induce changes in primary metabolism of plants through photosynthetic efficiency34, and elevated [CO2] may not increase wheat rusts incidence71, our results suggest that temperature was the main abiotic factor modulating the response of selected accessions against leaf rust infection. This may be due to plants prioritising their response to the most hampering abiotic stress47. Thus, it is feasible that elevated temperatures and [CO2] (environment 2), enhance those host physiological and molecular defence responses that are only expressed under elevated temperatures in the environment 175. In fact, the susceptible accession Qualidou was the only one presenting some parameters with statistical differences in S2 plants compared to S1 plants. A lower Total Pustule Area value was shown, probably due to a lower IF value and, more likely, due to the aforementioned enhanced host defence responses that limited fungal development and subsequent pustule formation, and sporulation potential71. Interestingly, this lower value did not significantly reduce the Total Disease Area, supporting the fact that S2 Qualidou plants expressed some enhanced defence responses against leaf rust disease, although this would ultimately reduce host photosynthetic area and yield14.Therefore, considering both elevated abiotic factors  during the inoculation process could reduce spore germination by up to 50%80. This reduction could lead a delay in the disease establishment, explaining the relevantly longer LP50 values in S1G and S2G plants of the Qualidou accession, as opposed to those in S1 and S2 plants. In addition, no significant differences of EA− and EA + values were observed in comparison with SB plants in any accession, indicating a reduction of infection sites in S1G and S2G plants prior to the establishment of host–pathogen infection. Furthermore, considering that elevated temperatures (30–35 °C) did not negatively affect the fungus once it had entered the host78, our results suggest that the inoculation process for S1G and S2G plants could also slightly weaken the subsequent disease progression. This could be observed in the reduced proportion of EST− in the Qualidou accession and EST+ in BL 28 and BL 38 accessions, together with lower CS values in the three selected accessions, all values compared to SB plants. However, these data were statistically relevant mainly in Qualidou accession and only for EST+ in BL 38 accession.Interestingly, the three selected accessions showed a significant reduction in leaf rust symptoms in S1G and S2G plants compared to SB plants, especially for the macroscopic parameters IF, Total Pustule Area, Total Disease Area, and Pustule Development Rate. In fact, based on empirical studies14. However, non-statistically significant differences in Total Pustule Area in Qualidou (susceptible) and BL 28 (moderately resistant) accessions were shown in S1G and S2G plants, suggesting that elevated [CO2] enhanced even more the host responses against leaf rust infection in S2G plants. This is particularly evident in the Qualidou accession, which also showed relevantly lower Mean Pustule Size and Pustule Development Rate values in comparison to S1G.Finally, S2G plants showed relevant differences in some macroscopic parameters compared to S1G plants, especially higher values of Total Disease Area for the three accessions, affecting more host photosynthetic area and reducing yield2] did not suppress the general defence response in our studied accessions BL 28 (moderately resistant) and BL 38 (resistant), nor did it cause the loss of susceptibility in Qualidou plants during P. triticina infection. This suggests that the genetic resistance background of these accessions was not temperature-sensitive83 or the timing and intensity of abiotic stresses were not sufficient to affect it84. Therefore, variations in macro and microscopic components of resistance in plants exposed to environments 1 and 2 were due to abiotic factors affecting durum wheat-leaf rust interactions mainly through modifications in the host and/or pathogen biology and physiology71. In contrast, leaf rust disease was greatly reduced when plants were inoculated and incubated under environments 1 and 2 (S1G and S2G plants), mimicking possible future heat events and disturbed rainfall patterns, suggesting that climate change would affect key stages of P. triticina and thus subsequent disease incidence in Mediterranean regions.In conclusion, the most important fact in our study is that elevated maximum temperatures alone or in combination with elevated [COSupplementary Tables.Supplementary Figure S1.


Moreover, vasoconstrictor use was related to death within 12 h of presentation and higher PvCO2. A large number of prospective studies should be performed to validate these results.Cardiogenic pulmonary edema (CPE) is the leading cause of cats visiting emergency hospitals with respiratory distress. The association of physical examination and venous blood gas parameters with the survival of cats with CPE was retrospectively investigated. Thirty-six cats were ultimately included, and eight of them died within 12 h after their presentation to our hospital. The Mann–Whitney U test with Bonferroni correction was used to compare the parameters. Cats that died within 12 h had significantly lower rectal temperatures and higher PvCO2 than those that did not die within 12 h. Moreover, hypotension and vasoconstrictor use were related to death within 12 h of presentation and higher PvCO2. These findings indicated the prognostic utility of body temperature and PvCO2, and the association between hypercapnia and the severity of CPE or hypotension. A large number of prospective studies should be performed to validate these results.Cats urgently visit emergency hospitals due to respiratory distress, and the chief cause is cardiogenic pulmonary edema (CPE). Although cats with CPE were frequently encountered in clinics, the prognostic factors were poorly reported. The objective of this retrospective study was to investigate the association of physical examination and venous blood gas parameters with the survival of cats with CPE in an emergency hospital. Thirty-six cats with CPE were ultimately included in the present study, and eight of them died within 12 h after their presentation to our hospital. Statistical analyses of clinical parameters between cats that died within 12 h and those that survived for 12 h were conducted using Mann–Whitney U test with Bonferroni correction. Cats that died within 12 h had significantly lower rectal temperatures and higher PvCO Congestive heart failure (CHF) is a life-threatening condition and a leading cause of emergency hospital visits for cats with respiratory distress . Cardiom2, HCO3−, and base excess (BE). The association between abnormal acid-base balance and mortality is reported in cats [2 >34 mmHg taken from unaffected limbs is associated with an increased risk of death [The use of blood gas analysis in veterinary medicine has increased recently , and it  in cats . Venous  in cats ,8,9. In 2 and clinical data, including body temperature, heart rate, respiratory rate, and blood pressure, in cats diagnosed with cardiomyopathy-induced CPE at an emergency hospital.While venous blood gas analysis has been assessed for some feline diseases, it has not, to date, been evaluated in cats with CHF, which is a common cause of feline respiratory distress. While feline CHF cases are frequently represented with CPE and/or pleural effusion, CPE is not curable with thoracentesis and needs more intensive care. Furthermore, any prognostic factors, which includes blood gas analysis, were analyzed focusing on cats with CPE not CHF. Therefore, this study aimed to identify the signalment or venous blood gas predictive factors for mortality and to evaluate the association between PvCO2 was measured if possible. As a “blood test(s)”, blood gas analysis, CBC, and/or biochemistry tests were selected by each veterinarian in charge. As an “ultrasonographic test”, point of care ultrasonography [Cats diagnosed with “CPE” at the TRVA Animal Medical Center between April 2019 and March 2021 were retrospectively included using a computerized search of the electronic medical records. The following exclusion criteria were used: (1) cats simultaneously diagnosed with ATE, (2) cats without blood gas analysis data within 1 h after their presentation, (3) cats treated using mechanical ventilation, and (4) cats without survival data after 12 h had passed after their presentation. The data from the first presentation were used if the cat had presented to the hospital multiple times with CPE during the included interval. At the hospital, when a dyspneic cat was admitted, immediate oxygen supplementation, physical exam, blood test, and thoracic radiographic and ultrasonographic tests were conducted, if acceptable for cats and their owners. As “physical exams”, body weight, body temperature, heart rate, and respiratory rate were measured in all patients. Non-invasive blood pressure or SpOnography ,11 was cA retrospective evaluation of clinical data using all medical records and any imaging studies (thoracic radiography and ultrasonography) was conducted by two clinicians to validate the diagnosis of CPE. The presence of radiographic evidence of cardiomegaly and regional or diffuse interstitial or alveolar opacity was used to help the diagnosis of CPE, as previously reported by Ward et al. . BecauseTreatment methods, drugs, the interval of hospitalization, and prognosis until discharge were retrospectively reviewed. Since the hospital is mainly for emergency patients during the night, we set 12 h as an interval to evaluate the short prognosis. Cats that died within 12 h were categorized as the “12-h death group” and those that survived beyond 12 h were categorized as the “12-h survival group”.2, HCO3−, base excess (BE), lactate (Lac), ionized calcium (iCa)). A heparinized syringe was used to conduct blood gas analysis using the Blood Gas Analyser Gem Premier 3500 .Among the clinical data on admission, body weight, body temperature, heart rate, respiratory rate, and blood pressure were recorded. Body temperatures below 32.0 °C were recorded as 32.0 °C since temperatures below this cannot be measured. Blood pressure was non-invasively measured using the oscillometric method. The data of jugular vein venous blood gas analysis with oxygen supplementation were also reviewed  curves were created, and cut-off points were calculated by optimizing the findings for specificity and sensitivity. Spearman correlation analyses were used to calculate the correlations between PvCO2 and heart rate, respiratory rate, and body temperature. Since blood pressure was not recorded in all cats, the correlation analysis was conducted in a smaller number of cats.Body temperature, heart rate, respiratory rate, the diagnoses of cardiomyopathies, and venous blood gas analysis parameters, including pH, PvCOThe owners of all participating cats gave consent for the future use of clinical data at their presentation. No animal was identifiable in this publication; therefore, additional informed consent for publication was not required.Eighty-three cats were diagnosed with CPE between April 2019 and March 2021. Among them, 20 cats diagnosed with ATE, 15 cats without survival data at 12 h, nine cats treated using mechanical ventilation, and three cats without blood gas examination data were excluded. Thus, 36 cats were included in the study, and their clinical data and CPE diagnosis were retrospectively reviewed. No cat with the original CPE diagnosis was judged to be incorrect. The 36 cats included mixed-breed (n = 20), Scottish fold (n = 6), American shorthair (n = 4), British shorthair (n = 2), Bombay (n = 1), Maine Coon (n = 1), Ragamuffin (n = 1), and Russian blue (n = 1). The median age of the 36 cats was 8 years . Twenty-one male cats (19 castrated) and 15 female cats (12 neutered) were included in the study. Eight of the 36 cats were previously diagnosed with cardiomyopathy and treated by primary care veterinarians. None of the 36 cats were previously diagnosed with CPE before their presentation to the hospital and prescribed diuretic drugs.The median body temperature, heart rate, respiratory rate, and body weight at presentation were 37.0 °C , 166/min , 86/min , and 4.3 kg , respectively. Systolic blood pressure was measured in 31 of 36 cats, and the median value was 122 mmHg . Among 31 cats whose blood pressure could be measured, four cats were considered to be hypotensive (<90 mmHg). Blood tests, ultrasonographic tests, and radiographic tests were performed with oxygen supplementation in 36, 36, and 35 cats, respectively. The results of venous blood gas analysis are shown in The proportion of cats that survived to discharge was 25/36 (69%). Euthanasia was not selected in any cats. Median hospitalization time for the surviving cats and for those that died was 35 h  and 10 h (2–60 h), respectively. Since the survival rate at 12 h was 78% (28 of 36 cats), twenty-eight cats were categorized in the 12 h survival group and eight in the 12 h death group.p-value = 0.01). The PvCO2 in the 12 h death group was also significantly higher than that in the 12 h survival group . Blood pressure was measured in 26 of 28 cats in the 12 h survival group and five of eight cats in the 12 h death group. Two of 26 cats in the 12 h survival group and two of five cats in the 12 h death group were hypotensive. Systolic blood pressure was significantly higher in the 12 h survival group . Vasoconstrictor agents, such as dopamine, norepinephrine, and vasopressin, were used in 12 of 36 cats. Vasoconstricting agents were used in six of eight cats in the 12 h death group and in six of 28 cats in the 12 h survival group . There was no significant difference among the diagnosis of cardiomyopathy between the two groups . Other data were not significantly different between the two groups of cats. Receiver operating characteristic (ROC) analysis was conducted using body temperature and PvCO2; the area under the ROC curve (AUC) was 0.862  for body temperature . The PvCO2 was significantly higher in cats treated with vasoconstrictor agents than in those not treated with the agents .Since PvCOassessed . A negatIn the present study, short-term prognostic factors in cats diagnosed with acute onset of cardiomyopathy-induced CPE were compared between cats that survived beyond 12 h and those that did not. Hypercapnia and hypothermia were identified as negative prognostic factors. Hypercapnia, as determined via venous blood gas analysis, may be an important prognostic factor for cats with CPE.Hypothermia was an adverse prognostic factor in cases of cardiomyopathy-induced CPE in this study, and body temperature less than 34.4 °C was suggested to be a negative prognostic factor for death within 12 h. Hypothermia has also been reported to be a negative prognostic factor in a previous study on feline CHF , which w2, that is, hypercapnia, was found to be a negative prognostic factor for CPE in cats in this study. A cut-off PvCO2 value of 52 mmHg might be able to predict cat death within 12 h. In a previous study, HCM cats with CHF showed significantly higher total venous CO2 in comparison with those diagnosed with ATE, syncope, or subclinical HCM [2, careful interpretation of PvCO2 is necessary especially in hypotensive cats.A higher value of PvCOical HCM . In thatical HCM ,21. In hical HCM . As in hical HCM . Since h2 and body temperature, between PvCO2 and heart rate, and between PvCO2 and systolic blood pressure were found. CHF is the situation in which there is decreased cardiac output and results in hypotension, hypothermia, and bradycardia [Negative correlations between PvCOdycardia . Since hdycardia , and hypdycardia , hypercaIn the present study, 69% of cats with cardiomyopathy-induced CPE survived to discharge; this proportion is lower than that reported by others in cats with CHF ,23. In aThere was no significant difference between 12 h survival and the diagnoses of cardiomyopathy. Since we included cats with CPE and excluded cats with ATE or pleural effusion, this result should be carefully interpreted. Long-term outcomes of feline CPE between different cardiomyopathies should be prospectively compared.p-value. Decreased iCa is frequently reported in critically ill cats and is considered to be the consequence of vitamin D deficiency or resistance, acquired or relative hypoparathyroidism, or hypomagnesemia [Ionized calcium was lower in the 12 h death group compared with the 12 h survival group with a nominal gnesemia . Calciumgnesemia . The cor2 ≥52 mmHg may be used to discriminate more severe cases. Careful interpretation of the present study is needed since we included cats with cardiomyopathy-induced CPE and did not include cats diagnosed with ATE, treated by mechanical ventilation, or without venous blood gas analysis or survival data at 12 h.In conclusion, hypothermia and hypercapnia were identified as negative prognostic factors in cats diagnosed with cardiomyopathy-induced CPE. Venous blood gas analysis may be a new prognostic tool in feline cases of pulmonary edema, and a PvCOThis study had some limitations. Due to the retrospective nature of the study, the cases did not involve a uniform treatment regimen. A prospective case–control study should be conducted to minimize the influence of differences in treatment procedures. Since only cats diagnosed with CPE were collected, some cats without the statement of CPE as a diagnosis might be missed from this search. Moreover, since the number of included cases in the present study was relatively small, a larger number of cases should be prospectively included in future studies. Blood pressure measurement and perfusion evaluation are required to evaluate the relationship between hypotension and hypercapnia. However, these evaluations could not be conducted in all included cases because of their clinical instability. Since we used a non-invasive oscillometric method to measure blood pressure, it was difficult to obtain precise values, especially in hypotensive patients. Venous blood gas analysis was not conducted in three cats within 1 h after their presentation in this study. The clinical instability of cats could influenced the decision in terms of venous blood gas analysis. However, since the three cats excluded because of the absence of venous blood gas analysis within one hour were discharged from the hospital (data not shown), they did not seem to be in a severe condition compared with the included cases. In the present study, prognostic factors of feline CPE at more than 12 h could not be evaluated. Since feline CPE occurs and progresses rapidly, the short-term outcome is important in emergency facilities. However, longer term outcomes should be prospectively examined to reevaluate prognostic factors. In the present study, the diagnosis of cardiomyopathy was conducted using echocardiographic data obtained by veterinarians working at emergency rescue not a cardiologist. Although the diagnosis was made by a cardiologist, the preserved ultrasonographic data might not be adequate to diagnose. Ultrasonographic data should be obtained and diagnosed by cardiologists in a future prospective study.