Xenostrobus securis  from variably contaminated sites (primarily from cadmium (Cd), chromium (Cr), copper (Cu), lead (Pb) and zinc (Zn)) in the highly urbanized Sydney Estuary, south-eastern Australia. Data were collected 15 years apart (June 2004 and June 2019) to assess any change in (i) the “health” of mussels (based on the above two toxicity endpoints) and (ii) their metal contaminant status . Linear relationships between both toxicity endpoints and metal concentrations in the whole soft tissue were also investigated. Multivariate statistical techniques, including principal components analysis, multidimensional scaling and cluster analysis, were also explored to reduce dimensional data, investigate patterns and assess similarities among study sites with respect to tissue metal concentrations and toxicity effects in X. securis. Enrichment factors were calculated by dividing the mean whole soft tissue metal concentration at each site in the Sydney Estuary, by its mean baseline metal concentration from near-pristine (reference) sites in the adjacent Hawkesbury Estuary. Salinity, pH, temperature, turbidity, dissolved oxygen and chlorophyll a were measured in the surface waters at each siteThis article contains a dataset of the genotoxic  and cytotoxic , via the neutral red retention test) effects on the pygmy mussel,  Specifications table This table needs re-formatting on the PROOF version•Data provide a quality-assured temporal assessment (2004 versus 2019) of the bioavailability and toxicity of key metal contaminants in the Sydney Estuary, whereby changes in metal contaminant status can be detected to better manage and protect marine ecosystems.•Data will be of benefit to coastal pollution researchers, managers, planners and policy makers, and ultimately, the public through greater recreational enjoyment of an iconic natural resource (Sydney Harbor waterways).•Xenostrobus securis – serving as a benchmark for further investigation.First genotoxic  and cytotoxic  field data worldwide for the estuarine (mytilid) mussel, •One of only few studies worldwide to report quantitative  field data for lysosomal membrane stability in mytilids.•X. securis.Data permit univariate and multivariate relationships to be determined among whole soft tissue concentrations of cadmium, chromium, copper, lead and zinc and toxicity effects  in 1a concentration) of surface waters in the Hawkesbury and Sydney estuaries, for both the 2004 and 2019 sampling events, is provided in Xenostrobus securis (pygmy mussel) from variably contaminated sites (1–24) in the Sydney Estuary, relative to near-pristine (reference) sites (R1−11) in the Hawkesbury Estuary, for the two sampling events (2004 and 2019), is presented in Variably contaminated sites 1‒24) in the highly urbanised Sydney Estuary, and near-pristine (reference) sites (R1‒11) in the adjacent Hawkesbury Estuary, in southern-eastern Australia, are presented in 4 in the X. securis as the dependent variable, are provided in Appendix A (Table S4 for ‰MF and Table S5 for %LMS). Inverse linear relationships between %LMS and ‰MF in the haemocytes of X. securis from the Sydney and Hawkesbury estuaries, for two sampling events (2004 and 2019), are presented in The results of factorial (two-way) analysis of variance, with site (1−24 and R1−11) and sampling time (2004 or 2019) as independent variables and (i) ‰MF and (ii) %LMS in the haemocytes of X. securis, from variably contaminated sites (1 − 24) in the Sydney Estuary, relative to reference sites (R1−11) in the Hawkesbury Estuary, for the 2004 and 2019 sampling events, are presented in X. securis from variably contaminated sites in the Sydney Estuary, are presented in r2) for Cd, Cr, Cu, Pb and Zn concentrations in the whole soft tissue of X. securis from the Sydney and Hawkesbury estuaries, as a function of ‰MF or %LMS, for two sampling events (2004 and 2019), are provided in X. securis and those in (i) filtered (0.2 µm) surface water, (ii) suspended sediments and (iii) surface sediment The whole soft tissue concentrations (µg/dry weight) of cadmium (Cd), chromium (Cr), copper (Cu), lead (Pb) and zinc (Zn), the five key contaminants in the Sydney Estuary X. securis, are given in Appendix A (Table S7). Dendrograms from agglomerative hierarchical cluster analysis, showing sites similarly grouped according to Cd, Cr, Cu, Pb and Zn tissue concentrations and toxicity effects (‰MF or %LMS) in X. securis, for the two sampling events (2004 and 2019), are presented in Two-dimensional principal component biplots, showing site scores (gray circles) and loadings (or correlations) of the environmental variables from variably-contaminated sites (1–24) in the Sydney Estuary and reference sites (R1−11) in the Hawkesbury Estuary, for the two sampling events (2004 and 2019), are presented in 22.12 (∼90% urbanized) and a length of 30 km. Tides are microtidal  and mixed semi-diurnal. The surface waters are generally well mixed because of tidal turbulence and low freshwater inputs. Twenty-four sites  (Lamarck 1819), resided in surface sediments [The Sydney Estuary , comprisur sites  were selX. securisFor over two centuries, much of the Sydney Estuary has been consistently exposed to chemical contaminants from urban and industrial sources. While much of the existing chemical contamination in the Sydney Estuary is from legacy industrial point sources, catchment run-off (via stormwater drains and sewer overflows) and submarine groundwater discharge, are current and ongoing sources of chemical contamination. Five metals – Cd, Cr, Cu, Pb and Zn – were identified as key chemical contaminants in the Sydney Estuary, based on mean enrichment in X. securis, a synonym of Limnoperna securis and L. fortunei kikuchii, is endemic to the intertidal zone of estuaries and coastal lagoons of southern Australia (range 22°10′−43°28′ S) and New Zealand, but has been introduced to southeast Asia  and southern Europe  in the last three decades (and now considered a problematic invasive species).2.2X. securis were sampled at each study site (−2/s) photoperiod.Wild populations of udy site  in June udy site , and cytudy site ). About udy site  and 2 unp >0.05) differences in MF, or LMS, between individuals (n = 10 per site) acclimated in the laboratory (for 36 h) and collected from the field .Following depuration, 20 mussels, comprising the largest 15% of the population  from each site, were assessed for (i) MF , (ii) LMX. securis were used to evaluate the genotoxicity and cytotoxicity of chemical contaminants  in the Sydney Estuary. Haemocytes, which form a large part of the haemolymph (or “blood”), are circulating single cells in an open vascular system that are exposed to contaminants that enter the body via food (digestive glands) and/or the water column (gills). They are involved in the circulation of oxygen and nutrients and play an important role in immune defense (e.g. phagocytosis) and the transport, accumulation (detoxification) and excretion of contaminants The haemocytes of 2.3Micronuclei formation provides a cumulative (time-integrated) and irreversible measure of chromosomal DNA damage (and genetic instability) caused by chromosome breakage or mis-segregation during mitosis 2HPO4, 2.7 mM KCl and 1.8 mM KH2PO4; pH 7.4), gently mixed in a siliconized Eppendorf tube (1.5 mL) and maintained at 4 °C. 80 µL of haemolymph suspension was gently spread on a glass microscope slide (pre-coated with 10% poly-l-lysine solution), placed in a dark humid chamber (17 °C) for 15 min for cells to adhere/settle, air-dried  for 10 min, fixed with 100% methanol for 10 min and air-dried for a further 10 min. Haemocytes were then stained (5% (v/v) Giesma solution) for 10 min, rinsed twice in washing solution (96.9 mg/L KH2PO4 and 109 mg/L NaPO4·2H2O) for 5 min each time, mounted (Eukitt) and air-dried overnight. Slides were then coded and randomized.For each mussel, 100 µL of haemolymph was extracted from the posterior adductor mussel sinus using a 1 mL sterile syringe (with a 22 gauge hypodermic needle), containing 100 µL of buffered saline  using a light microscope (Olympus BX50) at x1000 magnification. Micronuclei were identified according to the following criteria: (i) spherical or ovoid-shaped extra nuclear, non-refractory, bodies in the cytoplasm, (ii) diameter 1/3‒1/16 of the main nucleus, (iii) color, texture, and optical features resembling the main nucleus, and (iv) separate from the main nucleus (absence of chromatin bridge). The mean ‰MF was calculated using 20 mussels per site.Haemocyte viability was assessed using differential acridine orange/ethidium bromide (AO/EB) staining 2.4The LMS of haemocytes is widely recognised as a good indicator of cellular integrity  in marine mussels 4, 9.8 mM KCl, 9.8 mM CaCl2 and 20 nM 4-(2-hydroxyethyl)−1-piperazine-ethanesulfonic acid (HEPES); pH 7.4; salinity 30‰), gently mixed in a siliconized Eppendorf tube (1.5 mL) and maintained at 4 °C for up 20 min. 40 µL of haemolymph suspension was gently spread on a glass microscope slide (pre-coated with 10% poly-l-lysine solution), incubated in a dark humid chamber (17 °C) for 15 min for cells to adhere/settle and stained with 40 µL of neutral red (NR) solution  in 1 mL of dimethyl sulfoxide, 10 µL was added into 5 mL buffered saline) for 15 min. Slides were then coded and randomized.For each mussel, 100 µL of haemolymph was extracted from the posterior adductor mussel sinus using a 1 mL sterile syringe (with a 22 gauge hypodermic needle), containing 100 µL of buffered saline  was determined, using the scoring procedure developed by Martínez-Gómez et al. The toxicity (effect) endpoint used herein provides a quantitative measure (0–100%) of LMS, based on NR dye loss or lysosomal abnormalities 2.5The whole soft tissue of each mussel (20 per site) was carefully dissected from the shell . All solutions were prepared with deionised water . Procedural blanks were employed throughout mussel digestions and analyses to evaluate contamination. All analyses were corrected for blanks. A standard reference material  and sample duplicates were used to evaluate analytical accuracy and precision, respectively. The mean measured concentrations of Cd, Cr, Cu, Pb and Zn in the SRM were within their certified ranges. For duplicate samples and SRMs, the percentage coefficient of variation was typically 5 − 10%.2.6a, a proxy for pelagic (micro-) phytoplankton abundance (and an estimate of food levels for X. securis), in surface waters at each site was sampled and measured according to Markich and Jeffree Surface water (∼50 cm depth) at each site was measured in situ for salinity (‰), pH, temperature (°C), turbidity (NTU) and dissolved oxygen (% saturation), twice in June 2004 and 2019 during dry conditions, using a multiparameter water quality meter (YSI 6000UPG or Horiba U52). The pH was measured with a glass combined electrode calibrated using a tris/tris-HCl buffer  according to Del Valls and Dickson 2.7p ≤ 0.05) different 10) data. Significance was tested at the p = 0.05 level.Spatial (between sites) and temporal (between years) differences in (i) ‰MF, (ii) %LMS or (iii) whole soft tissue metal  concentrations were tested using factorial (two-way) analysis of variance. Where significant differences were detected, mean values were graphically compared, whereby non-overlapping 84% confidence intervals were deemed significantly  variance. The number of principal components was determined using a scree plot and/or parallel analysis (using the 95th percentile rule). Non-metric multidimensional scaling (nMDS), based on squared Euclidean distance on standardized (−1 to 1) data with 1000 random starts as the initial configuration  and/or Tuckers congruence coefficient. Hierarchical cluster analysis, based on Euclidean distance and unweighted average-linkage agglomeration on standardized (−1 to 1) data, was subsequently used to group similar sites on the nMDS ordination plots. Optimum cluster validity/quality was assessed using a combination of cluster indices  and visual dendrogram inspection.Multivariate statistical techniques were used to simultaneous examine several (dependent and independent) variables to reduce dimensionality, recognize patterns and group similar data. Principal components analysis (PCA), based on singular value decomposition of the correlation matrix, was used to compare similarities among sites based on metal  tissue concentrations and toxicity effects (‰MF and %LMS) in X. securis from a given site in the Sydney Estuary, was divided by its mean “background” concentration, pooled from mussels at 11 near-pristine sites in the adjacent Hawkesbury Estuary.An enrichment factor (EF) approach was also used to normalize and quantify the level of metal contamination, whereby the mean concentration of Cd, Cr, Cu, Pb or Zn in the whole soft tissue in a concentration in site surface waters, shoreline height for mussel collection, and photoperiod) and biological (mussel age and reproductive status) variables, and hence, maximize the capacity to discern the potential effects (geno- and cyto-toxicity and accumulation) of  contaminants on X. securis.This dataset was designed to minimize, or account for, the effects of key environmental (salinity, pH, temperature, turbidity, dissolved oxygen concentration and chlorophyll


Lentinula edodes) is commonly consumed worldwide and is cultivated in many farms in Korea using Chinese substrates owing to a lack of knowledge on how to prepare sawdust-based substrate blocks (bag cultivation). Consequently, issues related to the origin of the Korean or Chinese substrate used in shiitake mushrooms produced using bag cultivation have been reported. Here, we investigated differences in fatty acids (FAs) and stable isotope ratios (SIRs) in shiitake mushrooms cultivated using Korean and Chinese substrates under similar conditions  and depending on the harvesting cycle. The total FA level decreased significantly by 5.49 mg∙g−1 as the harvesting cycle increased (p < 0.0001); however, no differences were found in FAs between shiitake mushrooms cultivated using Korean and Chinese substrates. Linoleic acid was the most abundant FA, accounting for 77–81% of the total FAs during four harvesting cycles. Moreover, the SIRs differed significantly between the Korean and Chinese substrates, and the harvesting cycles resulted in smaller maximum differences in SIR values compared to those of the cultivation substrate origins. Our findings contribute to the identification of the geographical origin of shiitake mushrooms and may have potential applications in international shiitake-mushroom markets.Shiitake mushroom ( Lentinula edodes) is globally the second most commonly cultivated mushroom owing to its unique taste and flavor /Rstandard), where R is the C, N, O, or S SIR  of the shiitake mushroom samples and the international reference standards. Vienna PeeDee Belemnite, atmospheric N2, Vienna Standard Mean Ocean Water, and Vienna Canyon Diablo Troilite were used as the international reference standards for C, N, O, and S SIRs, respectively [13C, ±0.04‰ for δ15N, ±0.17‰ for δ18O, and ±0.29‰ for δ34S IRMS measurements.The final C, N, O, and S SIRs were reported in parts per thousand , compared with values of internationally established reference standards using the following formula: δ, ‰ = , and all data were analyzed using SAS software  with the general linear model. Least significant difference (LSD) tests with a probability level of 0.05 were used to determine differences among samples based on either the harvesting cycle or substrate origin. Furthermore, statistical analysis was conducted using two-way ANOVA with p-value for the main factor  and its interactions (H × S). Eight FAs—C16:0, C17:0, C17:1, C18:0, C18:1 n-9, C18:2 n-6, C18:3 n-6, and C24:0 were found in shiitake mushrooms; however, C17:0 and C18:3n-6 were observed at below the limit of detection (LOD) based on the signal-to-noise ratio (S/N = 3). Additionally, the level of total FAs decreased significantly by 5.49 mg∙g−1 as the harvesting cycle increased . In addition, UFA and total FA levels continually decreased in the shiitake mushrooms cultivated using Korean or Chinese substrate until the fourth harvesting cycle, whereas SFA levels did not change much after the second harvesting cycle  in each cycle ; desaturL. edodes, resulting in decreased palmitic acid but increased linoleic acid levels [A prior study has shown that low-temperature shock treatment of the substrate alters the lipid profile and accelerates the initiation of sporophores in d levels . Howeverd levels ,30,31. I13C, δ15N, δ18O, and δ34S in shiitake mushrooms cultivated using Korean and Chinese substrates at the same farm in Korea. The results of δ13C, δ15N, δ18O, and δ34S differed significantly with the Korean and Chinese substrates (p < 0.05), although SIR variations among the samples were mainly found with respect to the harvesting cycle. The maximum differences in the shiitake mushrooms according to the harvesting cycle were <1.0‰ for δ13C and δ15N, <2.0‰ for δ18O, and <3.0‰ for δ34S; these values were sufficiently small to discriminate the substrate origin. Only δ34S decreased as the harvesting cycle increased, and no clear trends were observed for δ13C, δ15N, or δ18O. Interestingly, shiitake mushroom samples cultivated using the Chinese substrate showed higher δ13C, δ18O, and δ34S compared with those cultivated using the Korean substrate; however, δ15N showed the opposite effect, with a higher value in shiitake mushrooms following cultivation using the Korean substrate (p < 0.05).13C is highly associated with plant photosynthesis type/efficiency, δ15N is related to local agricultural practices, δ18O is related to geoclimatic conditions, and δ34S is well reflected by geological features [13C, δ15N, δ18O, and δ34S compositions may differ in the substrate used for mushroom cultivation according to the mushroom farm, region, or country and could reflect their isotopic characteristics in the mushroom fruiting body [In fact, in the substrates, nutrient sources  are crucial components for optimal growth and for promoting mushroom growth because mushrooms cannot synthesize the nutrients required for their growth cycle . Additiofeatures ,34,35. Tfeatures ,37,38,39ing body ,24,40.13C, δ15N, δ18O, and δ34S in the dried shiitake samples enable clear discrimination and prediction of the geographical origin (when comparing China and Korea), with a probability of at least 98.7%. The isotopic features of shiitake mushrooms, which showed a higher mean value and wider distribution of δ34S, were consistent with those reported in the current study. Interestingly, despite the small sample sized-preliminary results of this study, the mean δ15N was higher in the shiitake mushrooms cultivated using the Korean substrate (3.02‰) than in those cultivated using the Chinese substrate (−1.33‰). This trend was similar to that reported previously [15N of the dried shiitake produced via mycelial cultivation from Japan (2.9‰) and Brazil (4.5‰) was higher than that produced from China (0.4‰). Thus, substrates can be a crucial reason underlying the δ15N difference in shiitake mushroom. Additionally, another study [13C, δ15N, δ18O, and δ34S differences in A. bisporus mushrooms in Korea. Despite the lack of detailed information on the cultivation substrate for A. bisporus mushroom, the difference in substrate components or their proportions used in a particular region or farm examined may be considered the most crucial factor in terms of the regional and farm-based SIR variations in the same A. bisporus cultivars .According to a previous study , δ13C, δeviously , whereiner study  reported13C, δ15N, δ18O, and δ34S variations in shiitake mushrooms cultivated using substrates. Moreover, the different origins of the substrates used resulted in variations in FA levels according to the harvesting cycle but showed nonsignificant variations in δ13C, δ15N, δ18O, and δ34S. These findings suggest that analyses of δ13C, δ15N, δ18O, and δ34S may be more useful than analyses of metabolites, such as FAs, for the identification of the geographical origin labelling of shiitake mushrooms.Our current study also reported the isotopic features and difference between shiitake fruiting bodies produced using two cultivation substrates; however, the SIR data for the cultivation substrates have not been included currently. Therefore, the SIR analysis of cultivation substrates together with more shiitake samples would be necessary to accurately confirm our current findings. This study is the first to report the weak effects of the harvesting cycle on δ13C, δ15N, δ18O, and δ34S in the shiitake mushrooms based on the substrate origin and the harvesting cycle. Eight FAs  were found in shiitake mushrooms, and linoleic acid was the most abundant FA, accounting for 77–81% of the total FAs during the four harvesting cycles. The UFA and total FA levels in shiitake mushrooms cultivated using Korean or Chinese substrate continually decreased until the fourth harvesting cycle, whereas SFA levels did not show major variations after the second harvesting cycle. The SIRs  differed significantly in the mushrooms cultivated using the Korean and Chinese substrates, and the maximum differences according to the harvesting cycle were <1.0‰ for δ13C and δ15N, <2.0‰ for δ18O, and <3.0‰ for δ34S. However, the SIR differences were sufficiently smaller to discriminate the substrate origin compared with differences in shiitake mushrooms grown using the Korean and Chinese substrates. Consequently, our findings verified differences in FAs and SIRs in shiitake mushrooms based on the substrate origin and harvesting cycle. Overall, our findings may provide important insights into recent issues related to the origin labeling of shiitake mushrooms produced in Korea by bag cultivation using Korean or Chinese substrate. However, further study should need to validate more clearly the effect of the cultivation substrates about FAs and SIRs levels along with more shiitake and substrate samples collected from both countries.In summary, our preliminary results showed variations and differences in FAs, δ


Following a re-analysis and validation of all analyses in this article, a very minor discrepancy in few numbers was uncovered, because in a power calculation for two-sample t-tests, (df+2)/2 was used in a formula instead of df+2.The third sentence of the abstract should read: “Median power to detect small, medium, and large effects was 0.12, 0.46, and 0.78, reflecting no improvement through the past half-century.”In the sixth paragraph of the results section, the third sentence should read: “For example, to detect a small true effect (d = 0.2), 90% of cognitive neuroscience records had power < 0.242.The first sentence of the tenth paragraph of the Results section should read: “The somewhat higher power in the journals we classified as more medically oriented was driven by the Journal of Psychiatry Research , which includes more behavioral studies than the other two journals we classified as ‘medical.’”th paragraph of the Results section, the fourth sentence on should read: “In the best case of having H0:H1 odds = 1:1 = 1 and zero bias, FRP is 13.0%. A 10% bias pushes this to 22%. Staying in the optimistic zone when every second to every sixth of hypotheses work out (1 ≤ H0:H1 odds ≥ 5) and with relatively modest 10%–30% experimenter bias, FRP is 22%–70% (median = 50%). That is, between one- to three-quarters of statistically significant results will be false positives. If we now move into the domain of slightly more exploratory research where even more experimental ideas are likely to be false , then FRP grows to at least 59%–90% (median = 75%).”In the 14Similarly, corrected power estimates in Please see corrected S1 Text(DOCX)Click here for additional data file.


The heat-tolerant variety BARI Gom 26 (“BG26”) maintained higher cellular homeostasis compared to the heat susceptible variety Pavon 76 (“Pavon”), perpetuated by higher accumulation of proline, glycine betaine, ascorbate-glutathione cycle associated enzymes, reduced glutathione and ascorbate concentration in plant cells. Significantly lower levels of MG detoxification and antioxidant activities and ascorbate-glutathione cycle-related enzymatic activities lead to increased susceptibility in variety “Pavon”. Hierarchical clustering and principal component analysis revealed that variety “BG26” possess a combination of biochemical responses tailoring antioxidant activities that induced a higher level of tolerance. Taken together, our results provide a pipeline for establishing a trade-off between antioxidant capacity and heat tolerance to facilitate functional genomics and translational research to unravel underlying mechanisms to better adapt wheat to heat stress.Heat stress alters photosynthetic components and the antioxidant scavenging system, negatively affecting plant growth and development. Plants overcome heat stress damage through an integrated network involving enzymatic and non-enzymatic antioxidants. This study aimed to assess physiological and biochemical responses in contrasting thermo-tolerant wheat varieties exposed to 25 °C (control) and 35 °C (heat stress), during the seedling stage. Our results revealed a substantial decrease in the photosynthetic pigments, carotenoids, anthocyanin content, and increased membrane injury index, malondialdehyde, methylglyoxal (MG), H After 5 days, two sets of seedlings (5 Petri plates for each variety with 20 seedlings in each plate) were then grown in two growth chambers with constant temperatures of 25 ± 1 °C (control) and 35 ± 1 °C (heat stress) with RH 75–80%, 16 h photoperiod with a light intensity of 200 μmol photon m−2 s−1 for 48 h. Growth chamber temperature and RH were monitored by a digital humidity and temperature meter . Petri plates were irrigated every day with half-strength Hoagland’s nutrient solution. Uniform sized seeds of four wheat varieties were selected and surface-sterilized with 70% ethanol followed by washing several times with sterile distilled water. The seeds were then soaked in distilled water for 10 min and sown in Petri plates (15 cm diameter) filled with sterile sand moistened with distilled water for germination for 3 days in the laboratory at a variable temperature of 26 to 32 °C. After germination, seedlings were then moved to controlled environment chamber maintained at 25 ± 1 °C during day and night, relative humidity (RH) of 75–80%, 16 h of photoperiod with a light intensity of 200 μmol photon mAfter termination of heat stress, shoot and root length and dry matter was recorded. Shoot and root length was measured from the root-shoot junction to the tip of the longest leaf and root, respectively. Dry matter was measured after oven drying the shoot and root at 60 °C for at least 2 days, till a constant weight was achieved. Before harvesting fully expanded leaves were collected into liquid nitrogen and used for pigment determination and biochemical assays. Expanded leaves from about 20 seedlings were collected to form a single replicate and the same repeated twice from an independent set of seedlings to obtain three biological replicates. All the measurements and assays were done in triplicate. Average of three biological replicates are presented in Tables and Figures.v/v) using pre-cooled pestle and mortar and the homogenate was centrifuged at 5000× g for 10 min. The absorbance was measured with a UV-visible spectrophotometer  at 663 and 645 nm and Chl contents were calculated using the equations proposed by Arnon   , where Xward.D2) . The prion 4.0.2 ,68. CorrIn the experimental setup including four wheat varieties and two growing conditions, all traits were significantly affected by both these factors but with different magnitudes . VariatiHeat stress substantially reduced shoot length (SL) and root length (RL) of all varieties . CompareThe Chl concentration in the wheat leaves decreased markedly under heat stress compared to control conditions in all varieties . CompareIn this study, leaf temperature depression (LTD) in all wheat varieties was decreased under heat stress (35 °C) conditions compared to control (25 °C) A. Among The membrane lipid peroxidation levels in leaf tissues measured as the MDA content increased significantly in seedlings exposed to heat stress, irrespective of the wheat variety A. Under The LOX activity of wheat leaves recorded a significant increase in all tested varieties, upon exposure to heat stress B. As a r2O2 level was observed in wheat leaves in response to heat stress compared to control  content, heat stress resulted in a substantial increase in GB content in all wheat varieties, compared to control B. GB conSimilarly, glutathione (GSH) content was significantly increased under heat stress in all four tested wheat varieties C. On expIn response to heat stress, an increase in SOD activity was observed in all wheat varieties with a significant increase in “BG23”, “BG25” and “BG26”, but not in “Pavon” A. Heat sThe CAT activity was significantly decreased under heat stress in all the wheat varieties B, recordSimilarly, POD activity was decreased non-significantly under heat stress in all wheat varieties C. Upon eHeat stress resulted in a significant increase in GPX activity in wheat seedlings of “BG26” but the increase was not significant in “BG23”, “BG25” and “Pavon” A. The hiThere was a significant increase in the glutathione reductase (GR) activity in all wheat varieties on exposure to heat stress A and theCompared to the control, the activities of DHAR and MDHAR was decreased markedly in all wheat varieties due to heat stress C,D. Upon2O2, MII, LOX, MG, “Chl a” and Chl a/b which formed Group-1  were obtained involving various physiological and biochemical variables measured among the wheat varieties A. Group- Group-1 A. In com Group-1 A. The ot Group-1 A. In add Group-1 A. “BG26”2O2, MDA, LOX, MG and MII were strongly associated with “Pavon”  or a cooler leaf has been used as a powerful and robust selection criterion to improve tolerance of plants to heat and drought stress ,80,81,822O2 is a toxic compound which is harmful to cells, resulting in lipid peroxidation and membrane injury [2O2, MDA and LOX levels in “Pavon” adds further support to a higher level of heat stress sensitivity, due to the inhibition or insufficient induction of the antioxidant defense systems compared to the tolerant “BG26”. MII, H2O2, MG, MDA and LOX collectively induce oxidative stress as these variables were significantly positively correlated under heat stress  in protecting some enzymes and protein complexes from heat-induced destabilization ,95 and i systems .2•−, which is then converted to H2O2 for subsequent metabolism to H2O by catalases (CAT) and peroxidases , thus protecting the cell damage [2O2 induced by heat. However, the extent of reduction varied with the relative tolerance of the varieties, i.e., comparatively higher reduction in susceptible “Pavon” than tolerant “BG26”  provides primary protection against Ol damage ,96. In tt “BG26” A–C. The e in SOD  and redue in SOD ,100,101 e in SOD  and maize in SOD .2O2 and lipid hydroperoxides (ROOHs)  are a family of isozymes that use GSH to reduce H (ROOHs) , and then et al. . The plan et al. . More tht stress A,C. SimiTaken together, apart from the AsA-GSH cycle, SOD, CAT, POD, GPX and GST are important antioxidant enzymes in plants. Our results demonstrated that the heat-tolerant “BG26” seedlings countered ROS production by maintaining a relatively higher amount of CAT and POD, and increasing the activities of SOD, GPX and GST under heat stress .2O2 detoxification (2O2 detoxification, these enzymes are also actively involved in the regeneration of non-enzymatic antioxidants like AsA and GSH. The presence of AsA and GSH has been reported to improve osmoregulation, plant water status and nutrient status, water use efficiency, photosynthetic performance, reduce oxidative stress and improve overall productivity in plants [Ascorbate–glutathione cycle is the core metabolic pathway to detoxify ROS and recycling of non-enzymatic antioxidants. This vital cycle contains four enzymes: APX, MDHAR, DHAR and GR which are systematically and proportionately involved in the Hfication . Other tn plants ,109. OurThe central role of GSH in the antioxidant defense system is due to its ability to regenerate AsA through reduction of DHA in the AsA-GSH cycle ,38 Figu. Our res2O2 to water (2O2 in chloroplasts, where CAT is absent, has been well established [2O2 scavenging [APX is a key enzyme in the AsA-GSH cycle and plays a vital role in plant defense against oxidative stress by catalyzing the conversion of Hto water . Its essablished . Our resablished , Almeselablished  and Tiwaablished . In addiavenging .Our results demonstrated that the activities of MDHAR and DHAR decreased markedly due to heat stress, which are partially supported by Rivero et al. . The sli2O2, MDA and MG content and reduced oxidative stress with enhanced antioxidant capacity. Our findings demonstrated that heat-tolerant attributes are closely associated with the overall antioxidant activities to aid plants to maintain cellular homeostasis under heat stress. Our results establish a reference for further molecular analysis in wheat at the seedling stage, shaping the antioxidant system associated biochemical responses aimed to better adapt wheat to early-stage heat stress. Further investigations are needed to establish a pipeline for translating findings from seedling stage to different developmental stages including yield and yield-related parameters.In conclusion, a comprehensive analysis of the oxidative species and antioxidant interactions allowed us to capture the pathways that induced greater heat tolerance in “BG26” seedlings exposed to heat stress, compared to other varieties. The “BG26” was able to maintain highest dry matter and pigment stability, due to increased proline and glycine betaine levels, lower accumulation of H


Although evidence-based interventions for increasing exercise among cancer survivors (CSs) exist, little is known about factors  that increase effectiveness and reach of such interventions, especially in rural settings. Such factors can be used to design implementation strategies. Hence, our study purpose was to (1) obtain multilevel perspectives on improving participation in and implementation of a multicomponent exercise behavior change intervention for rural women CSs and (2) identify factors important for understanding the context using the Consolidated Framework for Implementation Research (CFIR) for comparison across three levels .We conducted three nominal group technique meetings with rural women CSs, three with community/organizational stakeholders, and one with potential interventionists. During each meeting, participants were asked to respond silently to one question asking what would make a multicomponent exercise intervention doable from intervention participation (CSs) or implementation  perspectives. Responses were shared, discussed to clarify meaning, and prioritized by group vote. Data was deductively coded using CFIR.n = 19) was 61.8 ± 11.1 years, community stakeholders (n = 16) was 45.9 ± 8.1 years, and potential interventionists (n = 7) was 41.7 ± 15.2 years. There was considerable consensus among CSs, potential interventionists, and stakeholders in terms of CFIR domains and constructs, e.g., “Design quality and packaging” (Innovation Characteristics), “Patients needs and resources” (Outer Setting), “Available resources” (Inner Setting), and “Engaging” (Process). However, participant-specific CFIR domains and constructs were also observed, e.g., CSs endorsed “Knowledge and beliefs about the intervention,” “Individual stage of change,” and “Self-efficacy” ; potential interventionists valued “Tension for change” (Inner Setting) and “Innovation participants” and “Key stakeholder” (Process); stakeholders emphasized “Goals and feedback” and “Network and communication” (Inner Setting), and “Planning” (Process). How the three participant levels conceptualized the CFIR constructs demonstrated both similarities and differences.Mean age of CSs (Multilevel input yielded diversity in type, relative priority, and conceptualization of implementation facilitators suggesting foci for future implementation strategy development and testing. Findings also reinforced the importance of multilevel implementation strategies for increasing exercise in an underserved, at-risk population. Contributions to the literatureExercise can reduce mortality and morbidity after cancer. Hence, identifying implementation strategies is crucial for advancing the implementation science needed to translate exercise programs for cancer survivors into practice.Our findings identify and prioritize factors to be considered when designing strategies for successfully implementing an exercise program for rural cancer survivors from the perspective of three different types of stakeholders.Using the Consolidated Framework for Implementation Research (CFIR), our findings suggest differences in stakeholder perspectives that may guide strategy development.Our findings focus on an underserved population that suffers disparities related to health and physical inactivity.With technical advances in cancer diagnosis and treatment, cancer survivors (CSs) can live longer –3, and tThe Consolidated Framework for Implementation Research (CFIR), a comprehensive taxonomy that unifies and consolidates the array of constructs influencing implementation, has been used across a wide variety of study objectives and settings , 30. ItsGiven the need for exercise promotion among women CSs living in rural settings and the usefulness of CFIR, our study aims were as follows: (1) obtain multilevel perspectives  on improving participation in and implementation of a multicomponent exercise behavior change intervention for rural women CSs and (2) identify factors important for understanding the context using the CFIR for comparison across three levels . The intervention being translated is a 3-month program based on the social cognitive theory. It includes 12 supervised exercise sessions over a 6-week period that are tapered to entirely unsupervised exercise sessions during the final 6 weeks. Additional exercise education is provided by update exercise counseling sessions every 2 weeks during the home-based phase. Additional behavior change support is provided during six group discussions over the first 8 weeks that promote the development of a personalized behavioral modification plan .To identify potential factors that facilitate the implementation of a multicomponent exercise behavior change intervention  by non-rParticipants took part in nominal group technique (NGT) sessions. NGT is a well-established multi-step facilitated group meeting used with informant panels to elicit and prioritize responses to a specific question . The det= “What sorts of things would make delivering this program doable in this area?”.We prepared several candidate questions intended to elicit facilitators of participation in or implementation of an intervention that promotes exercise among women CSs in rural areas. After reviewing question accuracy and clarity, the final questions selected for the NGT panel meetings were (1) CSs = “What sorts of things would make this intervention doable for cancer survivors around here?” and (2) interventionists and stakeholders At the beginning of each NGT meeting, we briefly described the planned intervention and purpose of the meeting to participants. They were asked to silently generate and write their responses to the NGT question. Then, participants presented their responses to the group using a round-robin format. The responses were recorded on a flipchart. After the responses were exhausted, participants from each panel were given an opportunity to briefly discuss responses for clarification , to ensure that every response was understood from a common perspective. During these discussion phases, some responses were elaborated and a small number of responses were added. The final phase of each meeting consisted of a structured prioritization exercise that involved having each participant anonymously select from the group list what they individually perceived as the three most important things/factors from the group list and were not limited to selecting things/factors that they themselves nominated. Each participant was given a total of six votes and was asked to assign votes to the three things/factors based on perceived importance . The duration of each meeting varied, but on average was approximately 90 min. Weighted vote tally for a specific item was calculated by summing the weighted votes received . All weighted vote tallies were summed for a total for the group . The total vote amount was used as the denominator to calculate the percentage of total votes received by each factor; the percentage represents the consensus or agreement across participants in each NGT group.Descriptive statistics were conducted for the participant characteristics. Frequencies and percentages were calculated for categorical variables . Means and standard deviations were calculated for continuous variables . Fisher’s exact test was used for the analysis of the categorical variables due to small frequencies in the contingency tables. Analysis of variance, followed by the Tukey-Kramer multiple comparisons test for pairwise differences, was used for the analysis of the continuous variables. Statistical tests were two-sided and were performed using a significance level of 5%. SAS, Version 9.4 , and Tableau, Version 2018.3.2  were used for the statistical and visualization analyses.http://www.cfirguide.org/tools.html) by two research staff individually. Coding was iteratively discussed to reconcile coder differences. The percent of times a theme  was identified and calculated for CSs, interventionists, and stakeholders separately.The responses from each NGT meeting were entered and listed in tables. The individual rank orderings from each NGT meeting were aggregated across participants to tabulate a group level result. To broaden data utility, suggested facilitators were coded into the implementation science construct with the goal of using these constructs to guide the design of potentially useful implementation strategies. As such, the prioritized NGT responses were further coded based on CFIR domains and constructs for different participant types using the CFIR codebook (n = 19) was 61.8 ± 11.1 years (range 44 to 83 years). Nearly half of CSs reported a history of breast cancer (47.4%) with the remaining reporting bladder, lung, ovarian, uterine, melanoma, or leukemia; 57.9% were early stage; 78.9% were White; 42.1% reported annual income under $50,000; and the mean education level was 15.1 ± 2.7 years. The mean age of interventionists (n = 7) was 41.7 ± 15.2 years and stakeholders (n = 16) was 45.9 ± 8.1 years.Participant characteristics are displayed in Table p = 0.01), with greater income in the interventionist and stakeholder groups when compared to CSs. Given our focus on women CS, male representation was only present in the interventionist and stakeholder groups. There were also statistically significant differences between the mean age of the groups (p < 0.001) and mean education level of the groups (p = 0.019), with the mean age of the CSs being greater than that of the interventionists and that of the stakeholders, and the mean education level of the stakeholders being greater than that of the CSs.Statistically significant associations existed between income and group , accounting for 25.0% (9) of 36 total votes. Four participants assigned 8 votes to “Flexibility ,” accounting for 22.2% of total votes. Two participants assigned 3 votes to “Access to exercise specialist” (8.33%). One participant voted “Fee free for class, membership, and exercise specialist” (8.3%) as her most important. The top 4 factors for the CS panel 1 accounted for 63.9% of total votes  and “Flexible schedule/time for working/non-working survivors” (8.3%). One CS voted “Should be fun” (6.3%) as the most important factor. The top 4 voted factors took up 66.7% of the total votes (Table Thirteen of 42 responses were prioritized by CS panel 2. All participants in CS panel 2 assigned their votes to “Effective exercise” (for both beginner and advanced level) , accounting for 41.7% of total votes. Five of them voted “Effective exercise” as their most important factor and two as their 2nd most important factor. “Inexpensive costs, affordability” (13.3%) and “Facility with various equipment” (13.3%) were ranked equally important factors. Two CSs voted “Many forms of advertising” as important (10.0%). One CS assigned “Knowledge of self-care” (10.0%) and the other ranked “Staff and participants show caring” (10.0%) as most important. The top 6 factors received 83.3% overall group votes (Table Nine out of 18 responses were selected as important factors. Three participants in CS panel 3 endorsed “Convenient location” 26.7%) as either the most important or the 26.7% as eAmong 32 factors generated by the interventionist panel, 14 were endorsed and ranked. Four interventionists assigned 9 votes to “Financial support” (21.4%). Three interventionists assigned 5 votes to “Committed exercise specialist ” (11.9%). Four votes were assigned by two interventionists to “Set realistic/attainable goals for participants ” (9.5%). “Free transportation” was selected by two participants (7.1%). “Ensure demand for program” (7.1%), “Medical oversight and staff oversight” (7.1%), “Physician clearance” (7.1%), and “Educate referral source” (7.1%) were each endorsed as most important by one interventionist. The top 8 factors received 78.6% total group votes . “Promotion and awareness, e.g., newspaper, marketing, oncologist office, church, radio station, hospital website” received 4 votes from two panelists (11.1%). Two stakeholders ranked “Grant money or other resources to fund program, e.g., pay for refreshment, trainer, donation, operational costs” (8.3%) and “Training for start-up, train trainers” (8.3%) as most important, individually. The top 5 factors received 72.2% total votes . Three stakeholders assigned this factor as their most important and one stakeholder voted it as his/her 2nd most important votes from two stakeholders, respectively. One stakeholder assigned “Champion for program” (10.0%) as his/her most important. “Physician/Nursing staff engagement” (10.0%) received 3 votes from two participants. The first 5 factors accounted for 76.7% of total group votes . “Good communication tools” (13.3%) and “Dedicated team, e.g., personalized, whole structure of program” (13.3%) received the 2This stakeholder group generated 35 responses and endorsed and prioritized 10. Four of 5 stakeholders assigned 9 votes to “Physician buy-in (source of trust in program)” (30.0%). Three stakeholders gave 7 votes to “Transportation assistance for cancer survivors ” (23.3%). “Applying for funding to support staff, e.g., foundational, charitable” (10.0%) and “Pair participants with each other or other support person” (10.0%) were ranked as the most important. The top 4 factors received 73.3% of total votes (Table p > 0.05) relationship between the five major CFIR domains and participant type (Table The CFIR domains and constructs coded from the 223 NGT responses are aggregately depicted in Table pe Table . Participe Table  showed vnd largest CFIR construct for CSs was “Available resources” (10.4%) of Inner Setting domain. The 3rd largest CFIR construct for CSs was “Patient needs and resources” (8.3%) of Outer Setting domain, “Knowledge and beliefs about the intervention” (8.3%) of Characteristics of Individuals domain, and “Engaging” (8.3% for factors coded at the first level only for “Engaging”) of Process domain  of Inner Setting domain, and “Design quality and packaging” (12.5%) of Innovation Characteristics domain. Responses fell equally (9.4%) into “Patient needs and resources” of Outer Setting domain and factors coded at the first level only for “Engaging” of Process domain. Similarly, responses were equal (6.3%) in “Cost” of Innovation Characteristics domain, “Access to knowledge and information” of Inner Setting domain, and “Reflecting and evaluation” of Process domain. The remaining responses fell equally into 10 sub-categories in five CFIR domains  of Innovation Characteristics domain, “Available resources” 13.7%) of Inner Setting domain, and first level only for “Engaging” (11.6%) of Process domain. The next three large categories were “Cost” (7.4%) of Innovation Characteristics domain, “External change agents” (7.4%) of Process domain, and “Access to knowledge and information” of Inner Setting domain  , 54. SimStakeholders, a critical part of translating knowledge into action, can create a “user-friendly” environment or a network for CSs by providing different kinds of support for CSs or even interventionists . CommuniWith regard to external validity, the identified CFIR constructs in this report are similar to those from a recent study that focused on implementation of exercise interventions for both clinical and community settings . AlthougNotably, the data reported here also enhance our prior work in this setting. The results from the NGTs are similar to what we have reported from focus groups with interventionists and stakeholders . Of the There are some limitations that should be noted. First, all NGT participants were from a single region of a rural Southeastern US state and the identified factors potentially influencing implementation may not always be generalizable to other regions. However, many are indeed relevant  based on face value. Second, the majority of CS participants were White and, as such, results from this study might not well represent thoughts of CSs of other races. Third, half of CSs had a history of breast cancer; this might limit the usefulness of the data when implementing with individuals having a history of a cancer other than breast. These limitations are offset by several study strengths. We focused on an underserved and understudied population  while also addressing a critically important translational science area . Furthermore, we obtained multilevel perspectives on implementation and report novel data regarding the perceived relative importance of factors influencing implementation among different types of intervention “users.” We also used a formally facilitative approach, NGT, to identify potential users’ perspectives. Each NGT meeting was structured to promote equal involvement of all participants and minimize the process loss that arises from various non-task related interpersonal dynamics including extraneous and evaluative discussion that is often encountered in traditional focus groups and unstructured discussion sessions . Hence, Diverse factors potentially influencing implementation success for which implementation strategies could be developed and tested were identified. Such strategies could potentially influence the implementation of exercise programs for rural CSs, thus reducing an important health disparity. Clearly, the intervention design, cost, patient needs, available resources, and engagement are important across the stakeholder levels. However, the variability in the relative importance of the factors across the three levels supports directing strategies at the level for which it is most desired . The identified and prioritized factors anticipated to influence implementation also reinforce the importance of multilevel implementation strategies for increasing exercise in an underserved, at-risk population. It may be important for individuals involved in planning implementation strategies for exercise interventions to consider how different types of intervention users conceptualize and perceive the importance of CFIR constructs . Similarly, examining different stakeholders’ perspectives can be used to design for dissemination taking account each participant type’s perspective. Implementation toolkits for exercise programs for rural women CSs should include materials and guidance for addressing the identified factors potentially influencing implementation success. Further research is needed to determine optimal implementation strategies for addressing these factors and the mechanisms by which such strategies influence implementation outcomes through changes in CFIR constructs.


MTHFR C677T genotype were analyzed in 147 subjects with DS. For 77 subjects, metabolite levels were correlated with cognitive tests. Griffiths-III test was administered to 28 subjects (3.08–6.16 years) and WPPSI-III test was administered to 49 subjects (7.08–16.08 years). Significant correlations were found among some metabolite levels and between homocysteine levels and MTHFR C677T genotype. Moreover, homocysteine, UA and creatinine levels resulted increased with age. We did not find any correlation between metabolites and cognitive test score in the younger group. Homocysteine showed statistically significant correlation with WPPSI-III subtest scores when its level is ≥ 7.35 µmol/L, remaining correlated in higher thresholds only for non-verbal area scores. Vitamin B12 showed correlations with all WPPSI-III subtest scores when its level is < 442 pg/mL. The relevance of the present findings is the detection of a specific metabolite threshold related with a better or worse cognitive score, suggesting that vitamin B12 and homocysteine may have a role in cognitive development in children with DS.This work investigates the role of metabolite levels in the intellectual impairment of subjects with Down syndrome (DS). Homocysteine, folate, vitamin B12, uric acid (UA), creatinine levels and  Hsa21 gene expression in cells and tissues from subjects with DS has been reported as altered3 and recently associated with changes in the metabolic profile that might be involved in the development of the clinical features4. In particular, there are descriptions of Krebs cycle substrate modification5, one-carbon metabolism perturbation6 and hydrogen sulfide (H2S) over-production caused by an over-expression of the cystathionine-beta-synthase (CBS) gene7. The CBS gene, encoded by Hsa21, is over-expressed in trisomy 21 cells3 and permanently removes homocysteine (Hcy) and serine to obtain cystathionine and, subsequently, cysteine catalyzing a trans-sulfuration reaction8. CBS might also have a role in dysregulated DNA methylation levels in subjects with DS, causing decreased Hcy availability and consequentially methionine (Met) synthesis9.Down syndrome (DS) [MIM: 190685] is caused by an extra copy of allMTHFR C677T polymorphism  due to its well-known correlation with increased Hcy plasma levels10. 5-methyl-THF is the major donor of the methyl group, together with trimethylglycine (betaine) for the conversion of Hcy to methionine (Met)11. Met can be transformed into S-adenosyl-methionine (SAM) that, together with arginine and glycine, can be used for the production of creatine (precursor of creatinine). SAM is one of the principal methyl donors and when it loses its methyl group becomes S-adenosyl-homocysteine (SAH). SAH can regenerate Hcy and adenosine with the latter being converted into uric acid (UA). Hcy and folate metabolism are both parts of the one-carbon cycle, and because of this, they are closely related. In addition, the conversion of Hcy to Met is catalyzed by 5-methyltetrahydrofolate-homocysteine methyltransferase (MS), a B12-dependent enzyme that uses (5-methyl-THF) the derivative of folate as a methyl donor  since it catalyzes the conversion of 5,10-methylenetetrahydrofolate (THF) to 5-methyl-THF. In the present work, we detected 12. Subjects with DS resulted to have a lower tolerance to the antifolate drug methotrexate (MTX) and their blood levels of vitamin B12 and folate have been reported to be lower than healthy controls13.Hcy concentration has also been reported to be increased with low folate or vitamin B12 levels and lower with supplementation of vitamin B12 and folate14. Different studies show how Hcy and vitamin B12 could be involved in cognitive development. In a research study conducted in 2017, Murphy et al.15 noticed how an elevated maternal Hcy at preconception is associated with lower cognitive performance in children 4 months and 6 years after birth15 while vitamin B12 concentration was positively correlated with the cognitive score in 18 month old children16. ID is a constant feature in individuals with DS, even if it is present to an extremely variable degree17. Individual cognitive development is influenced by environmental factors20, but it may also involve biological mechanisms. Lejeune was the first who hypothesized that a “blocked” mechanism might determine the level of severity of ID21. Despite the individual variability, a specific cognitive profile of subjects with DS is reported in the literature: communication skills are generally found to pose the most significant challenges, non-verbal reasoning is a relative strength22, daily living skills are somewhat stronger, and socialization is the strongest adaptive skill25. The degree of ID changes over the lifespan of individuals with DS, in particular, their intelligence quotient (IQ) seems to decrease as they grow older due to their lower development rate with respect to typically developing children26. A previous study conducted in 2005 by Guéant and coll. on a cohort of 131 patients with trisomy 21 (mean age 20 years) reported how Hcy might influence cognitive development. They noticed how a lower IQ was significantly correlated with an Hcy level > 7.5 µmol/L (median) and an IQ < 40 was associated with Hcy concentration > 9.6 µmol/L (upper quartile)27.These results reinforce the idea that DS might be a metabolic disease due to the presence of biochemical alterations of fundamental pathways like the folate cycle, Hcy-Met cycle and Hcy trans-sulfurationMTHFR C677T polymorphism and Hcy plasma levels, the association among Hcy, folate, vitamin B12, UA and creatinine concentrations and their involvement in cognitive performance in subjects with DS.This work investigates the correlation between MTHFR C677T genotype and cognitive tests are listed in Supplementary Dataset A total of 147 children/young adults with a confirmed diagnosis of trisomy 21 and age > 2 years were studied . The information regarding age, sex, fasting state, the levels of Hcy, folate, vitamin B12, UA, creatinine, MTHFR C677T polymorphism were studied in all 147 subjects enrolled. PCR–RFLP analysis (detailed in the Methods section) divided the 147 subjects into three genotype groups: 40 CC (27.2%), 69 CT (46.9%) and 38 TT (25.9%). These groups resulted in being homogeneous for sex after contingency test (p = 0.339) and for age after Kruskal–Wallis test (p = 0.718), so we searched for a correlation between Hcy concentrations and MTHFR genotype.Hcy concentration level and MTHFR C677T genotype showing a significant difference in Hcy mean concentration (µm/L) among the three groups  (p = 0.045). Post-hoc analysis with Tukey’s test showed a significant difference in Hcy mean concentration between TT and CC genotypes (p = 0.035)  and fasting or non-fasting state p = 0.234) while a significant linear correlation with age at blood draw was found   and age at the time of blood sampling. In particular, we observed the increase of Hcy, UA and creatinine levels statistically correlated with the increase of age  correction, see Further analyses were assessed after removing strong outliers from fasting group. The statistical analysis reported two strong outliers  among the Hcy values (Hcy level ≥ 22.925 µmol/L) and two strong outliers  among the folate values (folate level ≥ 23.225 ng/mL). After the outlier removal, significantly moderate correlations were confirmed between Hcy and UA  and between UA and creatinine . Meanwhile, correlation strength between Hcy and creatinine decreased  and a new negative moderate correlation emerged between Hcy and folate  , verbal AE and IQ of WPPSI-III test with a weak negative correlation Table . VitaminStrong outliers were detected in the Griffiths-III group  and WPSSI-III group . Removing the outliers, no modification in statistical significance was detected among metabolite-cognitive correlations.27 for Hcy, folate, vitamin B12, UA and creatinine, we used the concentration values of 25th, 50th or 75th percentiles as references for dividing the subjects into two groups, one with a metabolite concentration value lower than the reference (Low or L group) and the other with a concentration value higher than or equal to the reference (High or H group) , we found no statistically significant correlation (Supplementary Table In the older group (7.08–16.08 years) the statistical analysis showed a moderate negative correlation between the H group of the 25th percentile value of Hcy  and the total AE scores , the non-verbal AE scores  and the IQ scores   and for Hcy ≥ 10.45 µmol/L, the H group of the 75th percentile,   showed a moderate positive correlation with verbal AE scores , non-verbal AE scores , total AE scores (r = 0.643 p = 0.003) and IQ scores (r = 0.572 p = 0.008)  no significative modifications occurred in correlations performed between metabolites percetile groups and cognitive data   in Antonaros et al.31, but none of them showed a correlation with IQ scores. These data suggest that metabolites analyzed in the present work could have a crucial role in cognitive function.In 50 of 77 subjects evaluated by Griffiths-III and WPPSI-III tests see  section,The first analysis performed in the older group including 49 subjects showed a weak negative significant correlation between Hcy levels and total AE , verbal AE  and IQ scores  . The negative association of Hcy levels with cognitive scores confirmed literature data in which the increase of Hcy plasma levels is reported as an early marker of cognitive impairment in the elderly population34 and as increased in psychiatric phenotype35. The relevance and originality of the present findings is the detection of a specific metabolite threshold related with a better or worse cognitive score, independent from the metabolite physiological range. In this view, a control group was not necessary to validate our data because a reference interval of the investigated metabolites has also been reported in the literature37.These analyses showed, in the older group, that the verbal area had a weak strength of significant correlation with the Hcy levels only in the H group,  and this is coherent with influences from different environmental variables  ” project  and metabolite dosage was detected by LUM.MTHFR C677T genotyping and plasma Hcy dosage, was collected in ethylenediaminetetraacetic acid (EDTA)-coated blood collection tubes and plasma fraction was isolated within two hours from blood collection42. Plasma fraction was stored at − 80 °C until it was sent to LUM of Maggiore Hospital, Bologna, Italy for Hcy plasma level detection. The rate of NADH conversion to NAD+ (measured at A340 nm) is directly proportional to the concentration of homocysteine, and it was detected by an automated chemistry analyzer AU 400 Beckman Coulter (https://www.axis-shield.com/wp-content/uploads/2019/01/FHRWAU-FHRW100-200-100_Generic-HCY-IFU_EN_Ver201601.pdf).The first aliquot, used for https://www.beckmancoulter.com/download/file/phxB03898L-EN_US/B03898L?type=pdf) and vitamin B12 (https://www.beckmancoulter.com/download/file/phxA89094L-EN_US/A89094L?type=pdf) levels in human serum was obtained by chemiluminescent immunoassay by Beckman Coulter Immunoassay Systems. The quantitative determination of UA in human serum was detected by enzymatic colour test by Beckman Coulter analysers (https://www.beckmancoulter.com/download/file/phxBLOSR6X9813-EN_US/BLOSR6X9813?type=pdf). The quantitative determination of creatinine in human serum was detected by kinetic color test (Jaffé method) by Beckman Coulter AU analyzers. The compensated method traceable to the isotope dilution mass spectrometry (IDMS) reference method was used (https://www.beckmancoulter.com/download/file/phxBLOSR6X7817-EN_US/BLOSR6X7817?type=pdf).The second aliquot was collected in EDTA free tubes and directly sent to LUM for routine blood analyses, which included folate, vitamin B12, UA and creatinine dosage. The quantitative determination of folate  reaction.31 and, in this study, they are indicated with the same DS patient code.Among the subjects enrolled who decided to participate in a cognitive evaluation, only the cognitive tests administrated within two months from blood sample collection were considered in order to correlate these data with metabolite levels. Thus, cognitive test results were available for 77 DS children out of the 147 subjects enrolled in the context of our clinical experimental study. Cognitive data of 50 subjects had already been shown in Antonaros et al.44. Depending on the age of the participant, the Griffiths-III test45 was administered to 28 subjects aged from 3.08 to 6.16 years, while the WPPSI-III test46 was administered to 49 subjects from 7.08 to 16.08 years. Griffiths-III is a play-oriented developmental test. It provides an overall measure of a child’s development across different areas: “foundations of learning” , “language and communication” , “eye and hand coordination” , “personal-social-emotional abilities”  and “gross motor” .Following a procedure increasingly more used in the field of ID and to have a more sensitive measure that avoided the floor effect, tests that were more appropriate for expected mental age rather than chronological were used to assess subject’s cognitive level46, consisting of different subtests, allows computation in three main indexes: verbal, non-verbal and total19. An IQ score was also calculated, by dividing the child's AE by his chronological age, multiplied by 100.For this study, scale A and scale B were considered. The former assesses critical aspects of learning during the early childhood years while the latter measures overall language development, including expressive language, receptive language, and, to a lesser extent, use of language to communicate socially with others . Raw scores were registered and transformed into AE scores which give us an idea of the level of development of any child in a given area, comparing that child with typically developing children of the same chronological age. The Wechsler Preschool and Primary Scale of Intelligence, third edition (WPPSI-III)MTHFR C677T genotype .Statistical analyses were carried out with SPSS Statistics . The p-value after FDR and Figs. Contingency tests were used to assess differences in sex among CC, CT and TT genotype groups, performed with SPSS Statistics software as follows: from the leading software Menu we selected “Analyze” and then “Descriptive statistics”, we then chose “Crosstabs”, we included Sex in “rows” and Genotype in “columns” boxes and finally selected “Chi-Square” in “Statistics” options. The Kruskal–Wallis test was used to assess differences in age distribution in Genotype groups , performed with SPSS Statistics software as follows: from the leading software Menu we selected “Analyze” and then “Non parametric tests”, we then chose “Independent samples”, we included Age in “tests field” and Genotype in “groups” boxes and finally selected “Run”.One-way ANOVA was used to assess differences in Hcy concentrations among the three genotypes. Analysis with the Tukey–Kramer method was performed as a post-hoc test for mean comparisons when ANOVA showed statistically significant results . These analyses were performed with SPSS Statistics software as follows: from the leading software Menu we selected “Analyze” and then “General linear model”, we then chose “Univariate”; we included Hcy data in the “dependent variable” box and genotype data in “fixed factor” box; finally we went to “post hoc” options and chose “Tuckey test” in the “equal variances assumed” section.The presence of outliers in metabolite level distribution was performed with SPSS Statistics software as follows: from the leading software Menu we selected “Analyze” and then “Descriptive statistics”, we then chose “Explore”, and included metabolite levels in “Dependent List”, finally in “Statistics section” we selected the “Outliers” and “Percentiles” options. SPSS Statistics software indicates strong outliers with an asterisk in the graph.A linear correlation was used to determine if the correlation between age and metabolite levels existed. It was performed with SPSS Statistics software as follows: from the leading software Menu, we selected “Analyze” and then “Correlate”, we then chose “Bivariate correlation”, and finally we included our data in the “variables” box.Correlations between sex and metabolite levels and between fasting/non-fasting state and metabolite levels were investigated with an unpaired t-test. One subject out of 147 without information about the fasting state was excluded from the statistical analysis. Unpaired t-test was performed with SPSS Statistics software as follows: from the leading software Menu we selected “Analyze” and then “Compare means”, we then chose “independent-samples T-test” and finally we included our data in the “test variables” box and inserted “Fasting/no fasting” in the “grouping variable” box.Partial correlation corrected by age at blood draw was used to correlate each metabolite level with all other metabolite concentrations. Only fasting subjects were included. FDR correction was applied to p-values obtained from these statistical analyses. Partial correlation corrected by age was performed with SPSS Statistics software as follows: from the leading software Menu we selected “Analyze” and then “Correlation”, we then chose “Partial correlation” and finally we included our data in the main box and inserted “Age at blood draw” in the “checked by” box). To obtain p-value after FDR correction we created a file with all the p-values obtained from the previous analysis and, using JMP software, from the main menu we selected “Add-in”, then “False Discovery Rate p-value” command and finally inserted the p-value column in “p value column”.27 as a model, the threshold concentration of Hcy, folate, vitamin B12, UA and creatinine levels were calculated using quartiles performing the following steps using SPSS software: from the leading software Menu we selected “Analyze” and then “Descriptive statistics”, we then chose “frequencies”, and finally we included our data in the “variables” box and chose “quartiles” in the “statistics” option. The concentration value of the 25th, 50th or 75th percentiles was used as a reference for dividing the subjects into two groups, one with a metabolite concentration value lower than the reference (Low or L group) and the other with a concentration value higher than or equal to the reference (High or H group) . Taking the study of Guéant et al.The study was approved by the independent Ethics Committee of the University Hospital St. Orsola-Malpighi Polyclinic, Bologna, Italy (study number: 39/2013/U/Tess). Informed written consent was obtained from all participants or, if participants are under 18, from a parent and/or legal guardian. The patients, if over 18, or their legal guardians were requested and required to sign an informed consent form for the collection of blood and clinical data before taking part in the study. All procedures were carried out in accordance with the Ethical Principles for Medical Research Involving Human Subjects of the Helsinki Declaration.Supplementary Dataset S1.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.


Objective: The study intended to use narrative medicine study for interdisciplinary collaboration to let medical and healthcare students have a chance to interact with one another and listen to patients’ stories to enhance students’ reflective thinking, communication, empathy, and narrative medicine writing skills. Methods: A fifteen-week quasi-experimental design was used to examine the learning outcomes of the intervention. Two groups of students were randomly assigned as the experimental group (33 students) and the control group (32 students). Before and after the intervention, both groups had to fill in a Reflective Thinking Scale for Healthcare Students and Providers (RTS-HSP), Patient–Healthcare Provider Communication Scale (P-HCS), Empathy Scale in Patient Care (ES-PC), and Analytic Narrative Medicine Writing Scoring Rubric (ANMWSR). Results: The findings showed that on the reflective thinking scale, experimental group students had significantly higher reflective thinking posttest scores in “reflective skepticism,” “empathetic reflection,” and “critical open-mindedness,” but not in “self-examination.” As for patient–healthcare provider communication, they had significantly higher posttest scores in all “perception of trust and receptivity,” “patient-centered information giving,” “rapport building,” and “facilitation of patient involvement” factors. As for empathy, they had significant higher posttest scores in “behavioral empathy” and “affective empathy,” but not in “intelligent empathy.” In narrative medical writing skills, they had significant higher posttest scores in the “attention → representation → affiliation,” “depth of reflection,” “focus and context structure,” and “ideas and elaboration” sections, but not in the “language and conventions” section. Conclusion: The findings suggest that narrative medicine is worth recommending for interdisciplinary collaboration for healthcare education. Overreliance on medical technologies results in an inhumane medical care system, causing healthcare professionals not to care much about the feelings and psychological experiences of patients. Patients, of course, prefer to have more interaction with healthcare professionals than impersonal technologies . HoweverAccording to Charon , narratiProviders who integrate narrative medicine into their practices can use self-reflection to examine their positions as physicians or professionals, endeavoring to research scientific objectivity to relieve patients’ pain and suffering . Charon Literature and narrative medicine share some similarities. Literature deals with suffering and dilemmas of all human beings; narrative medicine mainly deals with the suffering and dilemmas of patients, patients’ families, or healthcare practitioners . TherefoBoth literature and narrative medicine are the sharing and reflection of human experiences, particularly regarding suffering, dilemmas, desperation, or even dying . In “LitResearch ,18,19 haNarrative knowledge in literature study helps readers, through reflection, identify with others . TherefoTo facilitate healthcare communication among students and professionals, teachers may group learners considering their disciplines, thereby creating an interdisciplinary collaborative learning class. In the interdisciplinary collaborative learning class, students with varied healthcare background knowledge, skills, and expertise can work together collaboratively to consider patients’ illnesses from different disciplinary perspectives .The use of narrative medicine for interdisciplinary collaboration helps each participant actively take responsibility to work with one another to reach the set goal . MoreoveThe aim of the research was to use narrative medicine for interdisciplinary collaboration to let students from different disciplines listen to illness narratives from different perspectives, through narrative medicine. To reach the goal, the study intended to examine the effectiveness of using narrative medicine for interdisciplinary collaboration in reflective thinking, patient–health provider communication, empathy, and narrative medicine writing skills. The proposed hypotheses were:Hypothesis 1.Medical and healthcare students using narrative medicine for interdisciplinary collaboration will have higher reflective thinking levels than those not using narrative medicine.Hypothesis 2.Medical and healthcare students using narrative medicine for interdisciplinary collaboration will have better patient–healthcare provider communication than those not using narrative medicine.Hypothesis 3.Medical and healthcare students using narrative medicine for interdisciplinary collaboration will show more empathy than those not using narrative medicine.Hypothesis 4.Medical and healthcare students using narrative medicine for interdisciplinary collaboration will have higher levels of narrative medical writing skills than those not using narrative medicine.SD = 0.39) were informed the research purposes to let them decide whether to participate in the study. However, in order to decrease the unexpected outcome as confounding effects, such as Hawthorne effect or John Henry effect, the students did not know which groups there were in. All 65 students were college students with varied majors related to medical and healthcare disciplines, such as medical laboratory and biotechnology, speech language pathology and audiology, medical imaging and radiological sciences, psychology, occupational therapy, physical therapy, medicine, dentistry, nursing, etc. They were first-year and second-year college students, hence having little or no clinical experience. In addition, with different majors, both group students had little or almost no interaction outside the class. The research was approved by Institutional Review Board of the Chung Shang Medical University Hospital (CSMUH No. 14149). The participants were informed about the research purpose. All data from participants were blinded and all data were identified using numbers in order to prevent bringing any potential harm to students.Two homogeneous and normally distributed classes that varied in medical and healthcare majors were randomly assigned as the experimental group (33 students) and the control group (32 students). Before participating in the research, all students , a Patient–Healthcare Provider Communication Scale (P-HCS), an Empathy Scale in Patient Care (ES-PC), and an Analytic Narrative Medicine Writing Scoring Rubric (ANMWSR) were administered to both groups, and the researchers collected, analyzed, and compared the test results. Based on these learning outcomes, the researchers used the cluster algorithm for interdisciplinary collaboration to evaluate the efficacy of study of literature for the experimental group in comparison with the control group. Both the experimental group and control group used techniques for interdisciplinary collaboration to investigate ethical or moral dilemmas in stories, novels, dramas, or films. However, the experimental group used ideas from narrative medicine for purposes of such collaborative study, while the control group did not. After intervention, the participants’ posttest results of RTS-HSP, P-HCS, ES-PC, and ANMWSR were compared to evaluate learning performances.c was the number of students in the cluster, i was the ith student in the cluster, j was the ratio score in the RTS-HSP, P-HCS, and ES-PC, and In reference to Liao and Wang’s  cluster The experiment lasted for 15 weeks. Both experimental and control group students attended two-hour class instruction per week and at least two hours of independent study and discussion forum each week. Both the experimental group and control group used techniques for interdisciplinary collaboration to investigate ethical or moral dilemmas in stories, novels, dramas, or films. In addition, they had to go to the E-learning learning website for discussion forum  for indeMore specifically, the experimental-group students were taught the fundamental stages of narrative medicine, as characterized by Rita Charon , to streIn order to measure students’ reflective thinking levels, the study used a 22-item Reflective Thinking Scale for Healthcare Students and Providers (RTS-HSP) . The scaA 26-item, nine-point Likert scale Patient–Healthcare Provider Communication Scale (P-HCS)  was usedThe Empathy Scale in Patient Care  was usedTo assess students’ levels of narrative medicine writing, an Analytic Narrative Medicine Writing Scoring Rubric  had beenp < 0.05).The researchers used SPSS 14.0  to analyze quantitative data, including one-way MANOVA  to compare multivariate sample means and one-way MANCOVA , with pretest scores as covariates to control for the initial group differences. The confidence level was set at 95%  = 0.574; p = 0.682) between the experimental and control groups. In addition, the pretest results also showed no significant differences between the experimental group  and the control group  on the reflective skepticism  = 1.386; p = 0.244), self-examination  = 0.709; p = 0.403), empathetic reflection  = 0.066; p = 0.798), and critical open-mindedness  = 1.209; p = 0.276).To test Hypothesis 1, using one-way MANOVA, the pretest results showed no overall significant difference  = 2.497; p = 0.053 > 0.05). However, a significant relatedness existed between the pretests and posttests in “reflective skepticism”  = 4.277; p = 0.004; p < 0.01), “self-examination”  = 6.689; p < 0.000), and “critical open-mindedness”  = 6.204; p < 0.000) sections . Nonetheless, in the “self-examination” factor, the experimental group students had a higher score (Mean = 46.64) than control group (Mean = 44.68), but not reaching significant difference (p = 0.121 > 0.05).While looking into the adjusted posttest means see , the resHypothesis 2.Medical and healthcare students using narrative medicine for interdisciplinary collaboration will have better patient–healthcare provider communication than those not using narrative medicine.F  = 0.697; p = 0.597) between the two groups. In addition, the results showed no significant differences between the means of the groups  on the “perception of trust and receptivity” = 2.183; p = 0.145), “patient-centered information giving”  = 1.231; p = 0.271), “rapport building”  = 1.342; p = 0.251), and “facilitation of patient involvement”  = 0.355; p = 0.553).To test Hypothesis 2, with the use of one-way MANOVA, the pretest results revealed no overall significant differences  = 10.019; p < 0.000), “patient-centered information giving”  = 5.990; p < 0.000), “rapport building”  = 26.708; p < 0.000), and “facilitation of patient involvement”  = 8.580; p < 0.000; see After 15-week intervention, the one-way MANCOVA results presented a significant relatedness between the pretest and posttest performances in “perception of trust and receptivity  than the means of the control group  = 0.524; p = 0.667) between the two groups. In addition, the results presented no significant differences between the means of the experimental group  and the means of the control group  in “behavioral empathy”  = 1.293; p = 0.260), “affective empathy”  = 0.998; p = 0.322), and “intellectual empathy”  = 0.514; p = 0.465).The pretest results showed that, via one-way MANOVA, there was no overall significant difference  = 2.006; p = 0.123 > 0.05) and “affective empathy”  = 1.582; p = 0.203 > 0.05). However, the results showed a significant relatedness between the pretest and posttest performance in “intellectual empathy”  = 4.366; p = 0.008 < 0.01; see After 15-week intervention, the results showed no significant relatedness between the pretest and posttest performances in “behavioral empathy” . Nonetheless, there was no significant difference between two groups in “intellectual empathy”  and the control group  = 0.695; p = 0.629) between the two groups. In addition, the pretest results showed no significant differences between the experimental group  and the control group  in “attention → representation → affiliation”  = 0.532; p = 0.468), “depth of reflection”  = 1.945; p = 0.168), “focus & context structure”  = 0.787; p = 0.379), “ideas & elaboration”  = 0.170; p = 0.682), and “language & conventions”  = 0.174; p = 0.678).The pretest results showed that via one-way MANOVA there was no overall significant difference  = 1.190; p = 0.326 > 0.05), “focus & context structure”  = 2.011; p = 0.092 > 0.05), “ideas & elaboration”  = 0.520; p = 0.760 > 0.05), and “language & conventions”  = 0.531; p = 0.752 > 0.05). However, there was a significant relatedness between the pretest and posttest in “attention → representation → affiliation”  = 2.503; p = 0.041 < 0.05; see After 15-week intervention, the one-way MANCOVA results showed no significant relatedness between the pretest and posttest performances in “depth of reflection” . Nonetheless, in “language and conventions,” there was no significant difference between these two groups  than the means of the control group (Means = 2.94 and 3.21, The study used narrative medicine for interdisciplinary collaboration to strengthen medical and healthcare students’ reflective thinking, patient–healthcare provider communication, empathy, and narrative medicine writing skills. The results reveal that after 15-week intervention, both group students get higher performances in reflective thinking, patient–healthcare provider communication, empathy, and narrative medicine writing skills. The research results thus suggest that interdisciplinary collaboration can have positive effects on students whether or not they are situated in narrative medicine. In addition, students situated in narrative medicine have higher performances in reflective thinking, patient–healthcare provider communication, empathy, and narrative medicine writing skills, though some areas do not show significant differences. Regarding reflective thinking, students using narrative medicine for interdisciplinary collaboration had significantly higher reflective thinking levels than those who did not use narrative medicine in “reflective skepticism,” “empathetic reflection,” and “critical open-mindedness.” The results imply that students who received narrative medicine study have more chances, listening to abundant illness stories, to discover the dilemmas patients, patients’ families, and healthcare professionals encounter. These illness stories also give students more time to reflect on widely accepted knowledge. Hence, in order to lessen patients’ suffering, students could justify patients’ situations and check the credibility of information sources to find optimal solutions for patients and their families. The results correspond with Karkabi, Wald, and Castel’s  study anHowever, the results showed no significant difference in the “self-examination” section, though the students who received narrative medicine study had higher scores than those who did not receive the study. It could be possible that students are still too young to use self-examination without impartial analysis to decipher what their behaviors, thoughts, and feelings mean .As for patient–healthcare provider communication, students using narrative medicine had significantly higher patient–healthcare provider communication than those not using narrative medicine in all “perception of trust and receptivity,” “patient-centered information giving,” “rapport building,” and “facilitation of patient involvement” factors. The results imply that students receiving narrative medicine study can use the narrative medicine techniques, such as attention→ representation → affiliation, to empathize with patients’ inner and psychological perspectives. Therefore, in rapport with patients, students are more willing to offer patient-centered information, involving patients in their own healthcare decision-making, thus maintaining smoother and more harmonious communication with them. The results correspond with Hojat, et al.’s  study, bStudents who received narrative medicine study also showed more empathy than those who did not receive the study in the “behavioral empathy” and “affective empathy” section. The results imply that students situated in narrative medicine study are more willing to listen to patients, using facial expressions and eye contact to make them feel respected, and are inclined to comfort patients and patients’ families, if needed. They may also feel sad when they see patients’ misfortune and suffering. The results correspond with Charon’s ,44 studiHowever, the analysis results showed no significant difference in “intelligent empathy.” It could be that intelligent empathy involves consciously putting oneself in the place of patients and seeing things from their perspectives without any criticism to understand their emotions and consequences . Hence, In addition, students receiving narrative medicine study reached a higher level of narrative medical writing skills than those who did not in the “attention → representation → affiliation,” “depth of reflection,” “focus and context structure,” and “ideas and elaboration” sections. The results imply that students receiving narrative medicine study can use the triad of “attention → representation → affiliation” and connect their personal feelings to write touching illness stories. Moreover, these students can analyze and contemplate patients’ situations from different perspectives to arouse readers’ empathy toward patients’ dilemmas and suffering. However, there was no significance in the “language and conventions” section. It could be because that “language and conventions” are the basic feature of effective writing communication, or because students in the study are medical university students, with above-average college entrance scores. Therefore, these students have less difficulty with spelling, punctuation, and sentence structure.The study proposed using narrative medicine for interdisciplinary collaboration to let medical and healthcare students from different disciplines interact with one another and listen to illness narratives from multiple perspectives to realize people’s dilemmas and suffering, further building connections with patients’ illnesses. The results show that the use of narrative medicine can have positive effects on students in self-reflective thinking, in patient–healthcare provider communication, in empathy, and in narrative medicine writing skills, though not showing significant differences in some subscales.The study intended to allow medical and healthcare students in different disciplines listen to diverse voices in illness narratives and let them reflect, empathize, and communicate with patients. The contribution of the study lies in that students with different disciplines can acquire and share narrative reading and writing with one another, using narrative medicine techniques, such as attention → representation → affiliation, to reflect upon illness narratives from multiple perspectives. Hence, they can realize people’s suffering and dilemmas, learn to empathize and communicate with patients, and care about patients’ inner worlds. There is another contribution in the development of interdisciplinary collaborative learning, clusters, without which a successful implementation of narrative medicine for interdisciplinary collaboration in the study would not have been possible.The study demonstrates a successful attempt to use narrative medicine for interdisciplinary collaboration. However, continued cross-disciplinary and interdisciplinary collaboration is needed to further verify the study. Future study may consider the interrelations among communication, reflective thinking, empathy, and narrative medicine writing levels.