In North America, long-term wet deposition has been monitored through two national networks: the Canadian Air and Precipitation Monitoring Network (CAPMoN) and the US National Atmospheric Deposition Program (NADP), for Canada and the USA, respectively. In order to assess the comparability of measurements from the two networks, collocated measurements have been made at two sites, one in each country, since 1986 . In this study, we compared the measurements from NADP and CAPMoN instrumentation at the collocated sites at the Pennsylvania State University (Penn State), USA, from 1989 to 2016, and Frelighsburg, Quebec, Canada, from 2002 to 2019. We also included in the study the collocated daily-vs-weekly measurements by the CAPMoN network during 1999–2001 and 2016–2017 in order to evaluate the differences in wet concentration of ions due to sampling frequency alone. The study serves as an extension to two previous CAPMoN-NADP inter-comparisons by Sirois et al.  and Wetherbee et al., in . At the Penn State University site, for 1986–2019, CAPMoN was higher than NADP for all ions, in terms of weekly concentration, precipitation-weighted annual mean concentration, and annual wet deposition. The precipitation-weighted annual mean concentrations were higher for SO42− (2%), NO3− (12%), NH4+ (16%), H+ (6%), and base cations and Cl− (11–15%). For annual wet deposition, CAPMoN was higher for SO4−2, NO3−, NH4+ and H+ (5–17%), and base cations and Cl− (12–17%) during 1986–2019. At the Frelighsburg site, NADP changed the sample collector in October 2011. For 2002–2011, the relative differences at the Frelighsburg site were positive and similar in magnitude to those at the Penn State site. For 2012–2019, the precipitation-weighted annual mean concentrations were 5–27% lower than NADP, except for H+, which was 23% higher. The change in sample collector by NADP had the largest effect on between-network biases. The comparisons of daily-vs-weekly measurements conducted by the CAPMoN network during 1999–2001 and 2016–2017 show that the weekly measurements were higher than the daily measurements by 1–3% for SO42−, NO3−, and NH4+; 3–9% for Ca2+, Mg2+, Na+, and Cl−; 10–24% for K+; and lower for H+ by 8–30% in terms of precipitation-weighted mean concentration. Thus, differences in sampling frequencies did not contribute to the systematically higher CAPMoN measurements. Understanding the biases in the data for these networks is important for interpretation of continental scale deposition models and transboundary comparison of wet deposition trends.Wet deposition monitoring is a critical part of the long-term monitoring of acid deposition, which aims to assess the ecological impact of anthropogenic emissions of SOThe online version contains supplementary material available at 10.1007/s10661-023-11771-z. Long-term and routine measurements of chemical components of precipitation are critical parts of the long-term monitoring of acid deposition, and have been carried out around the world, e.g., the European Monitoring and Evaluation Programme (EMEP) and the Acid Deposition Monitoring Network in East Asia (EANET) . NADP also operated the Atmospheric Integrated Research Monitoring Network (AIRMoN) during 1992–2019. Samples from the NTN are collected on a weekly basis, while samples from the AIRMoN were collected on a 24-h basis, starting from the onset of a precipitation event. The NADP samples are sent to the Central Analytical Laboratory (CAL), which was within the University of Illinois prior to 2018 and currently is at the Wisconsin State Laboratory of Hygiene (WSLH).The measurements of wet deposition by NADP go back as far as 1978 through the establishment of the National Trend Network (NTN). The NTN is the only network providing long-term measurements of chemical components in precipitation across the USA (The CAPMoN network has been operated by Environment and Climate Change Canada (ECCC) since 1983 . In order to assess the comparability of data from the two networks, collocated CAPMoN and NADP measurements have been made since 1986 at two sites, one in each country. The measurements were analyzed by Sirois et al.  was in a rural area of southwestern Quebec, 5 km from the Canada-US border . The instruments were situated on a grassy open field surrounded by mixed forest. In late 2001, the Sutton site was relocated by 15 km to Frelighsburg, Quebec . The Frelighsburg site is in a rural area with some agricultural land use, well away from influence of industrial and urban activities, as described by Wetherbee et al. , nitrate (NO3−), ammonium (NH4+), hydrogen ion (H+ from pH), calcium (Ca2+), magnesium (Mg2+), sodium (Na+), potassium (K+), and chloride (Cl−). Specific conductance is also measured by NADP. CAPMoN also analyzes samples for total nitrogen.CAPMoN and NADP samples are analyzed for nine major ions: sulfate . Flame atomic absorption spectrometry was used for Ca2+and Mg2+ until 2013, when they began to be analyzed by ICP-AES. Flow injection analysis (FIA) colorimetry is utilized for total nitrogen (TN) and NH4+, and electrometry for pH. Trace samples (<= 0.2 mm of precipitation depth) are not analyzed. Sample analysis is prioritized for analysis of anions first, followed by base cations, NH4+, pH, and TN. Non-trace samples with minimum pipette volume of 0.5 mL or 1 mL were diluted to 9 or 5 times dilution, respectively, enough for anion analysis only. Samples with enough volume for complete analytical analysis are also diluted when the samples look highly contaminated prior to analysis or when they are determined to be outside the calibration range. pH analysis is not performed on diluted samples. Data quality assurance and quality control are performed using the Research Data Management and Quality Assurance (RDMQ) system  is utilized for ClPrior to 2018, weekly composited NADP samples were collected in deionized water (DI)–cleaned, high-density polyethylene (HDPE) buckets and transferred to DI-cleaned, 1-L HDPE bottles for shipment to the NADP Central Analytical Laboratory (CAL) at the University of Illinois-Urbana/Champaign (UI). In 2018, the NADP and CAL moved from UI to the Wisconsin State Laboratory of Hygiene, University of Wisconsin-Madison (UW). The NADP transitioned to a bag-lined bucket sampling system in 2020, the data for which are from beyond the study period. N-CON Systems, Inc. Model ADS 00-120 (NCON) sample collectors replaced the Aerochem Metrics Model 301 (ACM) collectors in a subset of NADP sites since 2010 . Prior to 2016, samples were analyzed for pH and specific conductance upon receipt, then filtered for the remaining analyses. During the study period, anions were measured by IC, major cations by inductively coupled argon plasma optical emission spectrometry (ICPOES), ammonium by FIA, and electrometry for pH and specific conductance. At the start of the study period, pH and specific conductance were measured first, and then samples of trace quantity were diluted to suitable volumes for remaining analyses. This analytical priority changed in 2016, and the order became FIA > IC > ICPOES, and then pH > specific conductance if undiluted sample remained. In 2017, the CAL transitioned to a new IC method that uses a separation column and eluent that are both different from those used by the previous method.https://nadp.slh.wisc.edu/networks/national-trends-network/) or by request. The NADP data are internally quality assured  is the calculated Winsorized standard deviation.Modified median absolute deviation (MMAD)  different from zero in statistics, respectively.In computing MMAD, first the median of a list of values, which in this study is the difference of each paired CAPMoN-NADP weekly concentration, is found; secondly, this median is subtracted from each value of the list; finally, a new median is determined from the new list. A coefficient 1/0.6745 is applied to the new median so that the calculated MMAD is the same as the traditional standard deviation when the list of value follows a normal distribution.In comparing weekly concentration, both arithmetic mean and median value are calculated and presented in Tables 42−, NO3−, NH4+, and H+, the relative differences of the median ranged from 0.003 mg L−1 for H+ to 0.148 mg L−1 for NO3−. In units of microequivalents per liter (μeq L−1), it ranged from 1.37 μeq L−1 for SO42− to 3.02 μeq L−1 for H+. The relative differences were 3.7%, 8.8%, 16%, and 7.6% for SO42−, NO3−, NH4+, and H+, respectively. During the warm (cold) season, the corresponding relative differences were 1.9% (6.6%), 5.4% (12.8%), 13.6% (18.5%), and 5.6% (9.4%). The relative differences were smaller during the warm season than the cold season, especially for SO42−, NO3−, and H+. The relative standard deviations were between 24.0% for SO42−, and 35.1% for NH4+. The spread of the one-to-one scatter plots (not shown) is quantitatively described by the metrics of CoV. The CoV for each ion was much smaller than the relative standard deviation, ranging from 9.5% for SO42− to 18.3% for NH4+. The correlation coefficient was between 0.83 for NH4+ and 0.95 for SO42−. In general, there was a good correlation between CAPMoN and NADP for the weekly concentrations of SO42−, NO3−, NH4+, and H+. The correlation coefficients for the warm and cold seasons were comparable for SO42−, NO3−, and H+. However, the correlation coefficient for NH4+ was 0.92 for the cold season, higher than 0.76 for the warm season.The statistics for the Penn State site during 1986–2019 are shown in Table −, the median differences ranged from 0.002 mg L−1 for Na+ to 0.013 mg L−1 for Cl−. In units of μeq L−1, it ranged from 0.09 μeq L−1 for Na+ to 0.60 μeq L−1 for Ca2+. The relative differences were between 5.6% for Na+ and 21.1% for K+. The CoV was between 19.7% for Cl− and 44.9% for K+, indicating generally the one-to-one comparisons of weekly concentration were more scattered for base cations and Cl− than for SO42−, NO3−, NH4+ and H+. Except for K+, there was a strong correlation for the weekly concentration between CAPMoN and NADP for base cations and Cl−, with the correlation coefficient ranging from 0.84 to 0.91. For Ca2+, Cl−, and Na+, the correlation coefficients were higher during the warm season than the cold season. As shown in Table p-values were less than 0.05 for all ions indicating that the weekly concentrations from CAPMoN were statistically higher than those from NADP. Scatter plots of precipitation-weighted monthly mean concentrations, instead of weekly concentrations for better visualization, for the Penn State site are shown in Fig. 42− had the best agreement between the two networks. The spread of the one-to-one comparison for NO3−, NH4+, and H+ was small, but there was an obvious positive bias for each ion. The spread of the plot for K+ was largest among all ions, and the spreads for Ca2+, Mg2+, Cl−, and Na+ were comparable.For base cations and Cl42−; 5–10% for NO3−, H+, and Cl−; 10–16% for Ca2+, Mg2+, and NH4+; and 21% for K+. The correlation coefficients were greater than 0.9 for SO42−, NO3−, H+, and Cl−; between 0.8 and 0.9 for NH4+, Ca2+, Mg2+, and Na+; and less than 0.5 for K+. The between-network biases were statistically significant (p < 0.05) for all ions.Overall, the relative differences of the weekly concentrations between CAPMoN and NADP were less than 5% for SO42−, NO3−, NH4+, and H+ were lower during 2005–2019 than 1986–2019, due to emissions reductions of SO2 and NOx in the eastern US and Canada since 1990 , close to 10% for NO3− and H+, and between 10 and 17% for NH4+, Ca2+, Mg2+, K+, Na+, and Cl−. The correlation coefficients were generally lower during 2005–2019, and it was greater than 0.9 for H+ only. This could be due to the aging of the sample collectors, either CAPMoN or NADP collectors, or both. The between-network biases were statistically significant for all ions. The two networks agreed better during the warm season, with the average relative bias of 8.4% during the warm season  of weekly CAPMoN-vs-NADP concentrations was low for SO42−, NO3−, and NH4+, with CoV ranging from 11.5 to 18.2%. The spread was relatively high for H+, Ca2+, Cl−, Mg2+, and Na+, with CoV ranging from 27.0% for Na+ to 30.7% for Mg2+, and K+ had the highest spread with CoV of 49.4%. The biases between CAPMoN and NADP were not statistically significant for SO42−, Ca2+, Na+, and Cl−, but were statistically significant for other ions from the t-test (not shown). However, the biases were statistically significant from the Wilcoxon signed-rank test, as indicated by the p-values in Table 42−, NO3−, NH4+, and H+, with r of 0.93–0.96. For base cations and Cl−, r ranged from 0.75 to 0.85, except for K+ with r of 0.57. The correlation coefficients for the warm season were higher than the cold season (Table S.r = 0.94–0.98) except for K+.During 2002–2011, for all seasons, the relative differences were 3.0%, 6.3%, 9.3%, and 13.5% for SO42−, NO3−, H+, Ca2+, Cl−, and Na+, ranging from 3.0 to 9.3%; moderate for NH4+, Mg2+, and K+, from 13.3% for Mg2+ to 17.7% for K+.Overall, CAPMoN has higher weekly concentrations than NADP for 2002–2011 at Frelighsburg. The relative bias was small for SO+, of no difference for H+, and 20% higher for other ions. For the current inter-comparison of CAPMoN-vs-NADP, we found that the relative biases changed from positive values during 2002–2011 to negative values during 2012–2019 for all season and cold season except for H+ and K+  and positive bias for H+ (43.0%) were statistically significant. Comparing 2012–2019 to 2002–2011, the average correlation coefficient for all ions decreased from 0.91 to 0.75 during the warm season, and from 0.81 to 0.60 during the cold season.For 2012–2019, the NCON sampler was used as the NADP sample collector at Frelighsburg. The inter-comparisons of collocated measurements at 12 sites in the USA during 2009–2015 by Wetherbee  showed tp-value and tau, were used to identify if the trend was statistically significant and the strength of the trend. For SO42−, the relative bias before 1998 varied much more than after 1998. During 1986–1998, the relative bias ranged from −11.2% in 1993 to 12.0% in 1998, with a mean value of 1.8% and standard deviation of 6.9%. During 1999–2019, the relative differences of SO42− varied less (3.1 to 9.9%), with a mean value of 5.5% and a standard deviation of 2.1%. There was a moderate increasing trend in the relative bias during 1999–2019, mainly due to cold season biases during 2010–2019. The relative biases during the warm season for 1999–2019 were small, with a mean value of 2.8% ± 1.9% versus 9.6% ± 4.1% during the cold season. The trend of the relative bias of NO3− was not statistically significant with p = 0.99 for the cold season, p = 0.24 for the warm season, and p = 0.39 for all seasons. The mean and standard deviation annual relative biases for each season were 9.4% ± 3.2% , 3.4% ± 4.9% (warm), and 13.3% ± 4.9% (cold). The M-K test for the trend in the annual relative bias for NH4+ indicated no significant trends for 1986–2010 and 2011–2019. However, there was a clear decrease of the relative bias from 2008 to 2011. The mean relative bias values for 1986–2010 and 2011–2019 were 18.0% and 11.1%, respectively. Seasonally, there was a weak decreasing (p = 0.05) and a weak increasing trend (p = 0.09) of the relative bias for 1986–2010 and 2011–2019 during the cold seasons. The relative bias during the warm season (18.7%) was higher than the cold season (14.7%). The relative bias of H+ had a decreasing trend (p=0.02) for 1996–2005, an increasing trend (p=0.02) for 2005–2011, and no trend for 2011–2019 (p=0.9). The relative bias of H+ turned negative during 2004–2007 for all seasons, during 2004–2008 for the warm season, and was negative only in 2004 for the cold season. Overall, for the recent one to two decades, the measured weekly concentrations from CAPMoN were higher than NADP at Penn State for SO42− (5.5%) and NO3− (9.4%) during 1999–2019, and NH4+ (11.1%) during 2011–2019.Time series of relative bias for each year are shown in Fig. −, the relative biases had an increasing trend for Ca2+ and Mg2+ (p = 0.06–0.09), and a weak increasing trend for Cl− (p = 0.2) during 1986–1997. During 1998–2019, the trends were flat for Ca2+, Mg2+, and Cl−. There is a known bias in Na+ concentration data collected by NADP before 1998 due to sample contamination  lower than CAPMoN during 1986–1998. Relative biases were positive during all seasons for Ca2+, Mg2+, Cl−, and Na+ during 1998–2019, except for a few years for Ca2+ during the warm seasons, with the mean values for the relative bias varying from 10.5 to 16.0%, and the standard deviations from 4.2 to 5.5%. For 1998–2019, the mean relative bias during the warm season were 4.4–11.4% for Ca2+, Mg2+, Na+, and Cl−, much lower than the values of the cold season (16.3–25.0%). For K+, there was a decreasing trend in relative bias for 1986–1993, an increasing trend for 1993–1999, a decreasing trend for 1999–2005, and no trend for 2005–2019. The corresponding mean relative bias and standard deviation during 2005-2019 was 17.4% ± 5.1%.For base cations and Cl2+, Cl−, Mg2+, and Na+, and since 2005 for K+, the relative biases between CAPMoN and NADP were generally constant. The weekly concentrations measured by CAPMoN were 10.5 to 17.4% higher. The relative biases during the warm season were much lower than during the cold season.Overall, since 1998 for Ca42−, NO3−, and NH4+ were highly correlated to each other (R > 0.89) at the Frelighsburg site. The M-K test indicated no trends for SO42− and NO3−, and a weak decreasing trend for NH4+ during 2002–2011, which was prior to the precipitation sampler changed from the ACM collector to the NCON collector in October 2011. For 2012–2019, there was a decreasing trend in relative bias for NO3−, and there were no statistically significant trends for SO42− and NH4+. The relative biases have changed from generally positive to generally negative since the sampler was changed. During 2002–2011, the mean values of the relative biases were 4.0% (SO42−), 6.7% (NO3−), and 14.2% (NH4+), compared to −4.3% (SO42−), −4.0% (NO3−), and −4.0% (NH4+) during 2012–2019. For 2002–2019, the agreement between CAPMoN and NADP was very good during the warm season, with low relative biases of −2.3% (SO42−), −2.7% (NO3−), and −0.1% (NH4+). For the cold season, the relative biases were relatively larger: −10.5% (SO42−), −6.3% (NO3−), and −10.4% (NH4+). This indicates that when the new NCON collector was deployed at the Frelighsburg site, the previous positive biases changed to negative biases. The biases were relatively small for the warm season, but greater during the cold season. For H+, the trend of the relative bias was similar to that at Penn State: there was a decreasing trend for 2002–2005, and a significant (α=0.05) increasing trend for 2005–2019, especially during the warm season. However, during the cold season, there was no statistically significant (α=0.05) trend during 2008–2019.As shown in Fig. −, there were no statistically significant trends for Ca2+, Mg2+, and Cl− during 2002–2011 (p > 0.7) and for 2012–2019 (p > 0.3). For Na+, there was no trend in relative bias for 2002–2011, but there was a weak decreasing trend for 2012–2019. For K+, there was a decreasing trend in relative bias for 2002–2011, but then it was mostly constant for 2012–2019. Comparing the periods before and after the collector change , the mean values of the relative biases changed from positive (2002–2011) to negative (2012–2019) from 9.9 to −7.8% (Ca2+), 13.6 to −4.4% (Mg2+), 7.0 to −1.9% (Na+), and 4.4 to −1.9% (Cl−). For K+, the mean relative bias changed from 21.8 to 8.7%. However, during the warm season, only Ca2+ had a negative mean relative bias during 2012–2019 (−4.3%), and positive mean relative biases varied from 1.6 to 6.6% for Mg2+, K+, Na+, and Cl−. Overall, with the NCON collector, the agreement between CAPMoN and NADP for base cations and Cl− was much better than the previous ACM sampler during the warm season, but for the cold season, except for K+, the relative biases changed from positive values to negative values and the absolute values remained relatively large.For base cations and Cl42− had a good agreement between CAPMoN and NADP for both annual and seasonal precipitation-weighted mean concentrations, with relative biases ranging 2.1–3.8%, CoV ranging 3.5–8.3%, and r >= 0.98. The agreement was exceptionally good for the warm season after 2000. Data for NO3− and NH4+ indicated consistent positive biases over the 1986–2019 period, 11.8% and 15.6%, respectively. The relative bias was comparable between the warm and cold season for NH4+, but was much smaller during the warm season than the cold season for NO3−. The correlation coefficients were greater than 0.97 for NO3−, and around 0.90 for NH4+, for both warm and cold seasons. The CoV for NO3− was exceptionally small (5.3%) for the warm season, indicating the agreement between CAPMoN and NADP would be very good if the biases were eliminated. The statistical metrics in Tables + was good with respect to relative bias (6.2%), CoV (8.7%), and correlation (r=0.99). However, after examining the time series of annual and seasonal precipitation-weighted mean concentrations for H+  and the correlation coefficient (0.5).For base cations and Cl42− and 5.9% for H+, and ranged 13.5–19.0% for other ions . For base cations and Cl−, r ranged from 0.61 to 0.82. The relative biases for the cold season were higher than the warm season except for NH4+ and Mg2+, and the averages of the relative biases were 10.4% and 15.1% for the warm and cold seasons, respectively. The relative biases changed from positive values during 2002–2011 to negative values during 2012–2019 except for H+. During 2012–2019, the relative biases ranged from −5.5 to −3.9% for SO42−, NO3−, and NH4+ . There was no statistically significant bias between CAPMoN and NADP except for H+ during the warm season of 2012–2019. During the cold season of 2012–2019, excluding H+ and K+, the relative biases were −19.2 to −7.4% with an average of −14.1% , the mean correlation coefficient reduced to 0.44 during the cold season, comparing to 0.95 during the warm season. These results indicate that, with the implementation of the NCON collectors by NADP, the agreement between CAPMoN and NADP improved significantly during the warm season, but during the cold season, relatively large biases existed.For Frelighsburg, separate statistical analyses were done for 2002–2011 and 2012–2019 due to the change of the NADP sample collector. The summary of statistical analysis is presented in Tables ns Table . The CoVH+ r = 0.8–0.99. F42− (5.1%), NO3− (14.9%), NH4+ (17.2%), and H+ (8.8%) (Table S.R >= 0.95) for SO42−, NO3−, NH4+, and H+. For base cations and Cl−, annual depositions estimated from CAPMoN data were 12.4–17.1% higher than from NADP data. The corresponding correlations were high for Ca2+ and Cl− (r = 0.96), relatively high for Mg2+ (r = 0.91), and moderate for Na+ and K+ . As expected, since the deposition depends on both the precipitation-weighted mean annual concentrations and the precipitation depth, both of which had positive median biases (Tables + (10.4%) and NH4+ (14.4%). During the cold season, the relative biases of the annual deposition were 8.0% for SO42−, 14.0% for H+, and 16.7–30.8% for other ions. During 2005–2019, the relative biases were 8.4% for SO42−, and 14.0–22.3% for other ions. The means of the relative biases of all ions during the warm season (10.2%) were smaller than during the cold season (21.0%)  and cold (13.9%) seasons. Strong correlation of annual depositions were indicated for all ions during the warm season (r= 0.96–1.0), except for slightly weaker for K+ (r=0.84). Overall during 2012–2019, except for H+ and K+, the agreement of annual deposition estimates from CAPMoN and NADP were very good during the warm season, but there were negative biases during the cold season.The annual depositions estimated from CAPMoN data were higher than from NADP data during 2002–2011 for all ions with a range of 11.1 to 23.0% Table S.g. The wa42−, NO3−, and NH4+. Relatively larger differences occurred for higher weekly concentrations for all ions. These higher weekly concentrations were usually associated with relatively lower weekly precipitation amounts . Table +, the weekly concentrations from the weekly measurements were higher than those from the daily measurements. Mean daily-minus-weekly concentration differences between the two sampling protocols were approximately −0.2 μeq L−1 for Mg2+, K+, Na+, Cl−; −0.4 to −0.8 μeq L−1 for NO3−, Ca2+, and NH4+; and −1.0 μeq L−1 for SO42−. The relative differences in precipitation-weighted concentration ranged from −1.2 to −3.1% for SO42−, NO3−, and NH4+; −4.2 to −7.2% for Ca2+, Mg2+, Na+, and Cl−; and −24.4% for K+. For H+, the weekly measurements underestimated the precipitation-weighted mean concentration by 2.5 μeq L−1, or 8.2%. The measurements from EW1 had relatively large biases, compared to the measurements from EW2, EG1, and EG2. If we only look at the relative differences between EW2 and EG1 or EG2, the weekly measurements overestimate SO42−, NO3−, and NH4+ by 1–2%; Ca2+, Mg2+, Na+, and Cl− by 3–5%; and K+ by 14%.From the spring of 1999 to the spring of 2001, a field study was carried out at Egbert, Ontario, Canada, by the CAPMoN network. Four identical CAPMoN precipitation collectors were deployed at the site to measure wet deposition of the common CAPMoN and NADP ions. Two samplers were operated to collect daily samples (coded as EG1 and EG2) and the other two collected weekly samples (coded as EW1 and EW2). The main objective of the study was to assess the comparability of the daily and weekly sampling. For this analysis, weekly precipitation-weighted mean concentrations were calculated from the daily sample data  for comparison to the corresponding weekly sample concentrations (coded as EW1 and EW2), analogous to the NADP comparison methodology. From the weekly concentrations, precipitation-weighted mean concentration was calculated for the entire study period 1999–2001, using the equation similar to Eq. 2+ had a relative difference greater than 1%. For EW1-vs-EW2 comparisons, only K+ had a relative difference greater than 1%. Using median value as a metrics, the weekly measurements showed 1–3% higher SO42−, NO3−, and NH4+ concentrations; Ca2+, Mg2+, Na+, and Cl− by 1–5%; and K+ by 6% compared to weekly composite values derived with daily measurements. Only H+ was higher in the daily composites, by 5%. Daily-vs-daily and weekly-vs-weekly comparisons are usually mentioned as “within-method” comparison, while daily-vs-weekly comparisons are named as “between-method” comparison. The results in Table S.Table S.+. Very good agreements of weekly concentration were found for SO42−, NO3−, and NH4+ between daily and weekly sampling. Tables +, and an increase in the precipitation-weighted mean concentrations for other ions. The effect on the precipitation-weighted mean concentration of H+ was −1.3 μeq L−1, or −30%, on average, while the effect on SO42−, NO3−, and NH4+ was less than 0.3 μeq L−1, or 3%. The effect on Ca2+ and Mg2+ ranged 0.3–0.6 μeq L−1, or 5–10%; the impact on Na+ and Cl− was approximately 0.2 μeq L−1, or 2%, and the impact on K+ was smallest in absolute value, being 0.1 μeq L−1, but 10.3% in relative difference as the mean concentration of K+ was the lowest among all ions.To aid the CAPMoN network in the transition of CAPMoN measurements of wet deposition at some sites from a daily basis to a weekly basis, parallel measurements with daily and weekly sampling frequencies were carried out by the CAPMoN network during 2016–2017 at Egbert and Algoma sites in Ontario, Canada, and at the Jackson site in Nova Scotia, Canada. Two types of sample collectors, C300 and D400, were deployed at Egbert. Only C300 samplers were deployed at Algoma and Jackson. As shown in the scatter plots  sites were not due to the difference in sampling frequency between the two networks, because CAPMoN weekly composite measurements from daily data were lower than the CAPMoN weekly measurements except for H+. Inter-comparisons of wet deposition measurements with different sampling periods, namely, weekly-vs-event, have been conducted in previous studies  and summer months (June–August) of the 1999–2001 and 2016–2017 data. The relative biases, expressed as daily-minus-weekly, of the precipitation-weighted mean concentration of H+ were reduced from 8.2% for the summer season to 1.1% for the winter season for 1999–2001 data. For the 2016–2017 data, the relative biases were reduced from 61% (for C300 samplers) and 79% (for D400 samplers) for the summer season to 23% (C300) and 21% (D400) at the Egbert site. For the Jackson site, it was reduced from 30 to 7.9%. No seasonal analysis was performed for the Algoma site due to limited data. Examples of scatter plots of weekly concentrations of H+ from daily samples vs. weekly samples are shown in Fig. + in precipitation samples when the overall acidity of precipitation decreased during the past three decades in eastern North America, which could account for larger discrepancies in the 2016-17 studies than in 1999-2001.Significantly lower concentrations of Ha et al.  and Sista et al. . It has a et al. . To test3−, NH4+, and H+.A number of potential causes for the observed between-network biases were proposed in Sirois et al. : (1) CAPWetherbee et al.  examined+, possibly due to a small amount of evaporation over the sampling week. However, the impact was only 1–3% for SO42−, NO3−, and NH4+, and it was in the opposite direction of the between-network biases. Additionally, Wetherbee and Rhodes  could be mainly due to the differences in sample collector performance for the two networks.The change of the comparability of sample depth between CAPMoN and NADP was consistent with the change of the between-network biases from positive to negative for most ions except for H+, measured by CAPMoN were higher than those from NADP when the ACM was deployed at the Penn State and Frelighsburg sites. With the NCON being deployed at Frelighsburg during 2012–2019, the sample depths from NADP and CAPMoN became comparable, and the overall between-network biases turned negative (except for H+ and K+). The between-network bias for H+ continued to be positive at Frelighsburg during 2012–2019. This is consistent with the results of the CAPMoN daily-vs-weekly inter-comparisons, which showed that the concentrations of H+ in weekly samples were substantially lower than daily samples. Biological uptakes/conversion and particle dissolution might have reduced the concentrations of H+ in weekly samples  for inter-network biases are laid out in the World Meteorological Organization Global Atmosphere Watch Manual for the Precipitation Chemistry Programme WMO, . The MMAWet deposition data from NADP and CAPMoN have been jointly used for evaluating global and regional models , NO3− (12%), NH4+ (16%), H+ (6%), and base cations and Cl− (11–15%). For annual deposition, CAPMoN was 5–17% higher for all ions, with the best agreement for SO42−.At the Penn State site, for the whole study period 1986–2019, CAPMoN was higher than NADP for all ions, in terms of weekly concentration, precipitation-weighted annual mean concentration, and annual deposition. The precipitation-weighted annual mean concentrations were higher for SO+, which was 23% higher; the NADP-measured precipitation amount was 3.6% higher. Correspondingly, the annual deposition from CAPMoN was 3–15% lower than NADP, except that the H+ deposition was 30% higher. This study further confirms that the implementation of a new type of sample collector introduced systematic and notable changes in measured concentrations and deposition within the NADP network, as indicated in the previous study by Wetherbee  and 14–23% (NH4+) higher than NADP when precipitation samples were collected by the ACM collector. However, when the NCON collector was deployed at the Frelighsburg site, the annual deposition of nitrogen by CAPMoN was 5% and 3% lower than NADP for NO3− and NH4+, respectively. This study shows that between-network differences and within-network differences  create biases in the data that need to be considered when assessing the impact on ecosystems from atmospheric deposition .The inter-comparison results at Penn State and Frelighsburg (for 2002–2011) show that the deposition of nitrogen measured by CAPMoN was 15–18% .In this study, we compared the weekly concentrations of nine major ions measured by NADP and CAPMoN at two sites located in the Penn State, USA, and Frelighsburg, Canada, for the periods of 1986–2019 and 2001–2019 respectively. The major findings from this inter-comparison study are summarized as follows:ESM 1(DOCX 312 kb)


The fear conditioning task in rodents serves as a valuable tool for studying learning and memory, as well as enhancing our understanding of post-traumatic stress disorder (PTSD) and other stress-related disorders. By utilizing controlled experimental paradigms, traumatic experiences can be mimicked in rodents, allowing investigation of underlying neurobiological mechanisms. During the fear conditioning task, animals learn to associate a neutral stimulus, such as a tone or a context, with an aversive, unconditioned stimulus, most often a mild foot shock. Then, the previously neutral stimulus becomes conditioned stimulus, and when the animals are re-exposed to it, they show fear response. The fear response is most commonly measured as the percentage of time spent freezing, which is defined as the time the animal spends motionless, except for cardiac and respiratory movement.While several brain regions are involved in processing and storing of these associative episodic-like memories, the spatial attributes rely primarily on the hippocampus, while emotional valence depends mostly on the amygdala Maren, . UnderstHartner and Schrader's article explores the effects of stress hormones, norepinephrine (NE), and glucocorticoids on inhibitory signaling in the Layer 2 of medial entorhinal cortex (MEC_LII) circuitry. The authors show the ability of NE to increase the frequency and amplitude of inhibitory inputs to the MEC, an effect that is cell-specific, mediated by the activation α1 adrenergic receptors, and modulated by co-administration of corticosteroids. This study sheds light on how stress influences underlying mechanisms of fear memory formation by demonstrating the ability of stress hormones to alter inhibitory synaptic inputs within the MEC-LII.Hippocampal formation and entorhinal cortex show a high degree of interconnectivity and functional interaction between the hippocampus and MEC is crucial for spatial memory processing  of the amygdala. As the amygdala plays a crucial role in fear conditioning  or conditioning context on its own, without the previously predictive unconditioned stimulus, such as mild foot shock. This leads to the formation of a new, inhibitory memory  and the ventral tegmental area (VTA) are key regions involved in emotional processing and assignment of valence (Namburi et al., The aim of this Research Topic is to offer New Insights into Synaptic Plasticity in Fear Conditioning. While previous studies on fear conditioning have mostly focused on examining the triad of brain regions crucial for fear conditioning -hippocampus, amygdala, and medial prefrontal cortex-, the articles in this Research Topic broaden the scope by investigating circuits and molecular mechanisms of other brain regions that play important role in fear conditioning and emotion processing. Additionally, this Research Topic is putting a spotlight on the inhibitory transmission, and the inhibition/excitation balance. The inhibitory transmission has been historically understudied. Albeit interneurons are fewer in number in areas crucial for fear conditioning they are not passive gain regulators but exert their robust inhibition via intricate network of connections on the excitatory projection neurons, thus shaping the network activity (Lucas and Clem, ACr: Writing—original draft. ACi: Writing—review and editing. AY: Writing—original draft.


In this work, new composite films were prepared by incorporating the disintegrated bacterial cellulose (BCd) nanofibers and cerium oxide nanoparticles into chitosan (CS) matrices. The influence of the amount of nanofillers on the structure and properties of the polymer composites and the specific features of the intermolecular interactions in the materials were determined. An increase in film stiffness was observed as a result of reinforcing the CS matrix with BCd nanofibers: the Young’s modulus increased from 4.55 to 6.3 GPa with the introduction of 5% BCd. A further increase in Young’s modulus of 6.7 GPa and a significant increase in film strength (22% increase in yield stress compared to the CS film) were observed when the BCd concentration was increased to 20%. The amount of nanosized ceria affected the structure of the composite, followed by a change in the hydrophilic properties and texture of the composite films. Increasing the amount of nanoceria to 8% significantly improved the biocompatibility of the films and their adhesion to the culture of mesenchymal stem cells. The obtained nanocomposite films combine a number of favorable properties , which allows us to recommend them for use as a matrix material for the culture of mesenchymal stem cells and wound dressings. Natural polysaccharides are promising materials for biomedical applications due to their biocompatibility, biodegradability, non-toxicity, low immunogenicity, and biological activity. The various polysaccharide-based polymer composites can be obtained in the form of films, sponges, electrospun mats, hydrogels, and nano- and microparticles. The design of polymer composites involves a combination of polymers with different chemical structures and physical forms, as well as a combination of polymers with nanoscale modifiers . PolymerCS is a polyaminosaccharide derived from the deacetylation of chitin; CS is biodegraded in the human body under the action of lysozyme to N-acetylglucosamine and glucosamine, natural metabolic products . CS has Komagataeibacter rhaeticus) [One of the most promising natural polymers for biomedical applications is BC, which is a linear, unbranched polysaccharide consisting of 1,4-glucopyranose units that is biosynthesized by microorganisms  ,11. BC iaeticus) . In BC, aeticus) ,14. Thisaeticus) ,18,19,20aeticus) . The introduction of BC nanofibers into CS matrices can lead to the formation of composite materials with a number of valuable properties, such as improved mechanical properties, an optimized surface charge, and useful morphological features. Both the starting material and the methods of BC nanofiber isolation can influence the efficiency of nanofibrillation and the properties of the products, including morphology and crystallinity, which further affect the physical properties of the material. BC nanofibers obtained by the mechanical treatment of BC are typically characterized by both long length and high aspect ratio . Since BC nanofibers have a large number of hydroxyl groups on their surface, and nanofibers isolated from BC have a high specific surface area, their surface can be easily functionalized and their interaction with the polymer matrix components can lead to a change in the polymer structure to form a nanocomposite with precisely tuned properties ,12,22,23Another important factor is the relationship between mechanical properties, geometry, and biological properties of scaffolds for tissue engineering. In this regard, the development of biocompatible composites containing nanoparticles is being considered ,24,25,26In our previous studies, we obtained BC-based composites by impregnating the squeezed BC gel film with polysaccharide solutions containing citrate-stabilized ceria nanoparticles (CeONPs)  and CS-bThe next phase of our research was to develop a multi-component CS-BC-CeONP composite. This composite would combine the advantages of both previously developed scaffolds. Thus, the aim of this study was to obtain CS-based nanocomposites containing ensembles of nanoparticles with different architectures (disintegrated BC nanofibers and ceria nanoparticles), to study their interactions and the influence of the amount of nanoparticles present in the composite to modulate the physical and mechanical properties and network architecture of the nanocomposite, and to improve its biomedical potential for MSC culture.−1 band appears and the 1430 cm−1 band changes  spectra of the original BC and disintegrated bacterial cellulose (BCd) a show thThe presence of carboxylate groups on the BCd surface is confirmed by the negative ζ-potential of the BCd dispersion (−15 mV). To study the interaction of BCd with CS, we titrated the aqueous dispersion of BCd with a solution of CS in 2% acetic acid and measured the ζ-potential of the resulting particles . The chaThe presence of these charges promotes the mutual repulsion of BCd nanofibers and prevents their aggregation during dispersion in water, which further ensures uniform distribution of BCd in the CS matrix.Cellulose is semicrystalline polymer with two different crystal structures in the native state, namely Iα and Iβ. Cellulose Iα crystallizes in a triclinic unit cell, space group P1 with a = 6.717 Å, b = 5.962 Å, fiber repeat c = 10.400 Å, α = 118.08°, β = 114.80°, γ = 80.37°, and Iβ in a monoclinic unit cell, space group P21 with a = 7.784 Å, b = 8.201 Å, fiber repeat c = 10.38 Å, γ = 96.5° .oclinic) . BecauseIt should be noted that the two WAXS patterns are similar to each other, but the X-ray diffraction peaks revealed for the BCd film have a lower intensity compared to those for BC. In other words, the intensity of the amorphous halo in the BCd pattern is much higher than that in the BC pattern. The reason is believed to be a partial amorphization  of BCd. It should also be noted that in the BCd film, an increase in the intensity of the (010) peak and a simultaneous disappearance of the observed . This meThe diffractogram of CS (The diffractogram of CS(80)-BCd(20)-CeONP(4) , (200), (220), and (311), respectively  [For CS(80)-BCd(20)-CeONP(4) and Cs(80)-BCd(20)-CeONP(8)  .A comparative study of swelling properties of the composite films  indicateb does not lead to a decrease in the strength of the nanocomposite film with increasing BCd content.To compare the contribution of both polysaccharides to the nanocomposite film properties, the mechanical properties of individual CS and BCd films were investigated . The BC-The incorporation of CeONPs into the polysaccharide matrices does not significantly change the Young’s modulus of the films formed . This faWe can also note that the incorporation of nanoparticles into the polysaccharide matrix, in both concentrations used in this work (4% and 8%), leads to a decrease in the ultimate deformation typical of polymer-inorganic nanocomposites.It is of special interest to characterize the mechanical properties of the developed nanocomposite material in the swollen equilibrium state. Indeed, for biomedical applications, film materials are used not in the dry but in the wet form. The mechanical tests of the swollen film CS(80)-BCd(20)-CeONP(8) with ~470% water relative to the weight of the dry material  showed t2 in the film. Taking into account the concentration of water in the material , the content of oxide nanoparticles is 8.1%.The thermal degradation of CS(80)-BCd(20) and CS(80)-BCd(20)-CeONP(8) in air proceeds in two stages a, which The thermo-oxidative degradation processes in the first, low-temperature step correspond to the initial stage of pyrolysis when dehydration, depolymerization, and decomposition of acetylated and deacetylated units occur simultaneously. At this stage, pyrolysis causes the random cleavage of glycosidic bonds, followed by further decomposition, resulting in the formation of C2, C3, and C6 fatty acids, including acetic acid and butyric acid. The further process of deep destruction of materials takes place at the high temperature stage, up to 500–550 °C. At this stage, the oligomeric and monomeric products formed at the first stage decompose in an oxygen-containing atmosphere resulting in the formation of gaseous substances . A similThe thermogravimetric analysis (TGA) results show that the incorporation of CeONPs into the polysaccharide matrix did not lead to a significant change in the temperature characteristics of the thermo-oxidative degradation of the material over a wide temperature range (up to 450–550 °C). In particular, the temperature resistance indices of CS-BCd and CS-BCd-CeONP films  were almThis trend is well illustrated by the differential thermal analysis (DTA) curves of the degradation processes of CS-BCd and CS-BCd-CeONP films b. The heThe CS film shows a uniform surface, while BCd shows a random network of dried nanofibers with a thickness of 20–30 nm . The surThe scanning electron microscopy (SEM) image of the CS film cryocleavage  shows noIn addition, the surface of the samples was examined using energy-dispersive X-ray spectroscopy (EDX), which allows the presence and distribution of chemical elements in a sample to be determined . EDX confirmed the incorporation of cerium-containing species into CS(80)-BCd(20)-CeONP(8). Cerium distribution maps showed the uniform distribution of this element in the samples .p < 0.05). In the other experimental groups, no significant differences in the number of nuclei were found compared to the control (p > 0.05). The number of nuclei on the surface of CS(80)-BCd(20)-CeONP(8) was significantly higher than in CS(95)-BC(5)-CeONP(4), while there were no significant differences with CS(80)-BCd(20)-CeONP(4) (p>0.05) (The number of isolated MSCs and cells in the flat colonies was significantly higher in CS(80)-BCd(20)-CeONP(8) compared to the control ((p>0.05) . p > 0.05). The spheroids in CS(95)-BCd(5)-CeONP(4) and CS(80)-BCd(20)-CeONP(8) were larger in size compared to the control and the other experimental groups (p < 0.05). In addition, there were no significant differences in the size of spheroidal colonies in control and CS(80)-BCd(20)-CeONP(4) (p > 0.05)  .On coverslips, the cells were evenly arranged and spread on the glass surface, forming a confluent/sub-confluent monolayer similar to syncytium, and had a typical elongated shape with multiple processes; in addition, some cells were in the process of division. Longitudinal linear structures stained red, actin microfilaments, were clearly seen in the cells.In all experimental groups, the cells were irregularly arranged on the surface of the samples in the form of single cells, flat, and spherical colonies . Some ofIn CS(80)-BCd(20)-CeONP(4) and CS(80)-BCd(20) (as a control), multiple separately arranged cells of both typical elongated shape and rounded/nearly rounded shape were detected . Flat stIn CS(95)-BCd(5)-CeONP(4), individually arranged cells of typical elongated shape and rounded/nearly rounded cells were observed in small numbers . Most ofIn CS(80)-BCd(20)-CeONP(8), the separately arranged cells of typical elongated shape and rounded/nearly rounded cells were detected in small numbers . Most ofTherefore, the greatest number of adherent cells located separately and as part of planar colonies (outside of spheroids) was detected on the surface of CS(80)-BCd(20)-CeONP(8). In addition, the number of isolated cells , including those in a state of apoptosis, was greater on the surface of both the control CS(80)-BCd(20) and CS(80)-BCd(20)-CeONP(4) samples. On the surface of CS(95)-BCd(5)-CeONP(4) and CS(80)-BCd(20)-CeONP(8), most adherent cells were found in flat and spheroidal colonies. Spheroidal colonies were observed in all samples. No significant differences were found in the number of spheroidal colonies on the surface of the samples, with the colonies in CS(95)-BCd(5)-CeONP(4) and CS(80)-BCd(20)-CeONP(8) being larger. Maximum cell migration was observed in CS(95)-BCd(5)-CeONP(4) and CS(80)-BCd(20)-CeONP(8). Finally, the proliferation of adherent cells with primary formation of squamous colonies was more pronounced in CS(80)-BCd(20)-CeONP(4). CS(80)-BCd(20)-CeONP(8) showed the best adhesion properties and biocompatibility.In summary, cell adhesion, spreading, proliferation, differentiation, and function are highly dependent on scaffold properties such as mechanical properties, chemical structure, nano- and microtopography, surface charge, porosity, wettability, etc. ,43. CS-bThe developed composite containing CS, BCd, and CeONPs showed an altered topography with a stronger interaction between CS, BCd nanofibers, and CeONPs. Apparently, the altered topography improved the adhesive properties of the composite scaffolds. The combination of complex physicochemical interactions, including hydrophobic, coulombic, and van der Waals forces between the cell membrane and the material surface, occurs at the very beginning of the cell adhesion process ,46. ThusThe CS sample  from crab shells with a molecular weight of 160,000 (capillary viscometry) and a degree of deacetylation of 0.80 (conductometric back titration) was used . Kommagateibacter xylinus  in aqueous solutions containing 2 wt% of glucose, 0.3 wt% of yeast extract, and 2 wt% of ethanol at 30 °C for 14–21 days [BC was produced by –21 days ,49. BCs The CS film (control) was prepared by extruding a 3% solution of CS in 2% acetic acid through a spinneret onto a glass substrate, followed by drying at room temperature and further heating at 80 °C for 4 h.The detailed protocol of BCd film formation has been described elsewhere .h) of 9 nm.CeONPs used in the experiments were synthesized according to the protocol described elsewhere . CeONPs h of 244 nm.CS-stabilized CeONPs were prepared according to the procedure described elsewhere . CS-coatThe film-casting mixtures were prepared by mixing a 2% solution of CS in 2% acetic acid, a 0.3% dispersion of BCd, and a dispersion of CeONPs. The resulting mixtures were homogenized by mechanical stirring for 1 h. Films were prepared from the composite mixtures by dry casting; the mixtures were extruded through a spinneret onto a glass substrate and dried at room temperature (film thickness was 30–40 μm). The composite films were then heated at 80 °C for 2 h, which caused them to lose their solubility in water . The comFTIR spectra of the films were recorded on a Vertex 70 IR Fourier spectrometer . A Pike MIRacle attenuated total reflection sampling accessory  with a ZnSe working element was used to preserve the structure of the films. In the registration of the spectra, a correction was introduced that takes into account the penetration depth depending on the wavelength.The ζ-potential of BCd nanofibers was determined using a Photocor Compact-Z instrument  at a laser wavelength of 659 nm and a detection angle of 90°.To study the interaction between BCd and CS, a solution of CS in 2% acetic acid (C = 0.48 mg/mL) was added dropwise to a dispersion of BCd  and the ζ-potential of the resulting systems was measured.The equilibrium swelling degree of the films (the swelling degree after 24 h exposure to water) was determined by the gravimetric method.The resulting films were characterized by SEM using a SUPRA-55VP scanning electron microscope  and by WAXS using a D8 DISCOVER X-ray diffractometer  with CuKα radiation.2 detector . The analysis was performed over the whole visible range of the samples.SEM images were obtained using both a secondary electron detector and a backscattered electron detector. To visualize the distribution of CeONPs in the samples, the films were frozen and split in liquid nitrogen, then glued to a conductive tape, sputtered through a thin layer of platinum, and analyzed EDX to obtain the maps of Ce distribution. The elemental maps were obtained using an EDX-Max 80 mmb), and ultimate strength (εb) were determined.An AG-100kNX Plus setup  operating in uniaxial extension mode was used to investigate the mechanical properties of the films. Strip-like samples (2 × 20 mm) were stretched at room temperature at a rate of 2 mm/min according to the requirements of ASTM D638. The stress–strain curves of the samples were recorded during the tests. Young’s modulus (E), yield stress . A DTG-60 thermal analyzer  was used and samples (~5 mg) were heated in air to 600 °C at a rate of 5 °C/min.Adhesive properties of the materials were investigated using MSCs derived from the adipose tissue of healthy donors. The study was conducted in accordance with the Declaration of Helsinki, and approval was obtained from the local Ethical Committee of the Almazov National Medical Research Centre . Written informed consent was obtained from all subjects prior to adipose tissue biopsy. The study was conducted as previously described ,42,52.2 incubator at 37 °C and 5% CO2.The biocompatibility of cell culture with nanocomposite samples was investigated using CS(80)-BC(20) and 12 mm diameter coverslips as controls. Cell culturing was performed in α-MEM medium supplemented with 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin/streptomycin solution in a CO2 incubator (to ensure uniform distribution of the components of the medium in the structure of the samples). After 24 h, the medium was removed and a suspension of MSCs at a concentration of 50,000 cells/mL was added to the wells and co-cultured for 72 h in a CO2 incubator. The experiment was performed in triplicate.Samples of 12 × 8 mm materials (depending on the size of the well) were kept in phosphate-buffered saline (PBS) with the addition of 2% penicillin/streptomycin solution for 30 min, followed by three washes with PBS. The coverslips were sterilized in 70% ethanol for 10 min, followed by three washes with PBS. The samples and coverslips were then placed in the wells of a 24-well plate. Then, 1 mL of medium was added to the wells and incubated for 24 h in a COAfter 3 days, the samples and coverslips were transferred to the wells of a new plate, washed with PBS, and fixed with 4% paraformaldehyde solution for 10 min. After fixation, samples and coverslips were washed from paraformaldehyde with PBS and stained with rhodamine-labeled phalloidin according to a previously developed protocol. According to the protocol, samples and coverslips containing cells were first treated with 0.05% Triton X-100 solution for 3 min and then washed three times with PBS. Next, a solution of rhodamine-conjugated phalloidin at a dilution of 1:500 in 1% fetal bovine serum solution in PBS was added to the wells, incubated for 20 min at room temperature, and then washed five times with PBS. Finally, cell nuclei were stained with DAPI  at a dilution of 1:40,000, incubated for 40 s, and then the samples were thoroughly washed from DAPI with PBS.After staining, the samples were stored in PBS in the dark at +4 °C. The coverslips containing cells from the control wells were mounted on glass slides with mounting medium and stored in the dark at room temperature.2), and estimating spheroid size . The number of nuclei in spheroids was not assessed because it is technically impossible to accurately count DAPI-positive signals in colonies with a 3D structure. The morphology of cells and their colonies was assessed qualitatively by staining the cytoskeleton, the type of colonies , the presence and extent of cell migration along the periphery of spheroid colonies . Statistical processing of the obtained data was performed with the GraphPad Prism 6.01 software  using the non-parametric Mann–Whitney U criterion. The results were presented as mean ± standard error.Fluorescence microscopy with qualitative and quantitative analysis of adherent cells was performed on the stained MSCs on film samples and glasses. An Axiovert inverted fluorescence microscope  and a compatible Canon camera were used to visualize the cells. Pieces of material containing cells were placed between two coverslips. DAPI fluorescence was recorded using the DAPI channel, and rhodamine-phalloidin fluorescence was recorded using the rhodamine channel. Ten different fields of view were photographed at ×100 and ×400 magnifications for each technical replicate. Quantitative analysis consisted of counting the number of nuclei on the surface of the film samples (separately located cells and cells in flat colonies in fields of view without spheroids), counting the number of spheroid colonies per unit area  on the structure and properties of the composites was investigated. An increase in film stiffness was observed as a result of reinforcing the CS matrix with BCd nanofibers: the Young’s modulus increased from 4.55 to 6.3 GPa with the introduction of 5% BCd. A further increase in Young’s modulus of 6.7 GPa and a significant increase in film strength (22% increase in yield stress compared to the CS film) were observed when the BCd concentration was increased to 20%. The incorporation of CeONPs into the polysaccharide matrices has no significant effect on the mechanical properties of the films, while the X-ray diffraction data indicate the interaction of CeONPs with the polymeric components of the composite and the nature of this interaction depends on the amount of nanoparticles introduced. The introduction of 4% CeONPs is accompanied by an increase in the hydrophilicity of the nanocomposite, an increase in the CeONP concentration up to 8% contributes to a change in the structural organization of the nanocomposite, accompanied by a decrease in the swelling degree and the formation of a denser structure. The CS(80)-BCd(20)-CeONP(8) composite exhibits good strength in both dry and swollen states.All nanocomposite matrices were found to be biocompatible. The CS(80)-BCd(20)-CeONP(8) composite showed the best biocompatibility and improved cell adhesion to the scaffold surface. Thus, the positive effect of the introduction of BCd and CeONPs on the properties of the CS-based nanocomposite film was demonstrated, and the resulting films were found to be promising for use as a matrix material for MSC culture. In addition, the results obtained suggest the possibility of successfully using the developed materials for the preparation of wound dressings.


Type 1 diabetes mellitus (T1DM) is a chronic and lifelong condition that requires adequate behavior management in order to meet desired health outcomes. The effects of T1DM on the neurocognitive functioning of affected individuals raise concerns about how the disease may influence executive functioning. Inhibition is a core component of executive functioning, and plays a vital role in self-regulation and the restriction of impulsive behaviors. Inhibition may thus play a vital role in the behavior management of people with T1DM. The aim of this study was to identify current gaps in existing knowledge regarding the relationship between T1DM, inhibition, and behavior management. This study employed a critical review design to analyze and synthesize the current scientific literature. Twelve studies were identified through an appraisal process, and the data extracted were thematically analyzed and integrated. The findings of this study indicate that a possible cycle arises between these three constructs, in which T1DM affects inhibition, inhibition affects behavior management, and poor behavior management affects inhibition. It is recommended that future research should focus more specifically on this relationship. The relationship between type 1 diabetes mellitus (T1DM), inhibition, and behavioral management remains under-researched.Twelve studies were identified through an appraisal process, and these were thematically analyzed and integrated.A vicious cycle arises as the results indicate that there is an interdependent relationship between T1DM, inhibition, and behavioral management.The current scientific literature explores the relationship between type 1 diabetes mellitus (T1DM) and executive functioning (EF); however, research exploring T1DM and inhibition specifically as they relate to behavior management is limited. Inhibition is important in the control and adjustment of behavior, emotion, and cognition, and, considering the complex demands of managing a condition such as T1DM, it is an important function to consider. T1DM is considered a severe, chronic, and lifelong condition, and commonly manifests as the presence of high blood sugar levels –4. GregoT1DM is severe and leads to various complications, such as neuropathy, loss of consciousness, seizures, cardiovascular difficulties, kidney failure, blindness, and neurocognitive difficulties –12. The One of the neurocognitive difficulties identified through research is difficulty in executive functioning , 18–27. Inhibition, according to Chung et al.  and GoldSwanson and Maltinsky  assert tThus far, the literature has predominantly placed the focus on the umbrella term, executive functioning, as it relates to T1DM and the behavioral management of people with T1DM. Moreover, the existing literature has mainly been concerned with a one-directional relationship: the influence of executive functioning on T1DM management. Limited scientific literature has placed the focus on inhibition, T1DM, and behavioral management. More specifically, research that examines the bidirectional relationship between inhibition, T1DM, and behavioral management is limited.According to the scientific literature, what conclusions can be drawn regarding inhibition, T1DM, and the management of T1DM?The findings of this study may provide valuable information and identify current gaps within existing knowledge to enable future research to adopt a more specific focus. Therefore, the proposed study aimed to answer the following research question: A critical review provided the primary researcher with the necessary framework to meet the study’s aims of evaluating and integrating the existing body of knowledge to provide an in-depth and holistic perspective on the phenomena . It is ohost  , ScienceDirect® (Elsevier), Google Scholar , Academic Search Premier (EBSCO Information Services), SocINDEX with Full Text (EBSCO Information Services), PsycArticles® , PsycInfo® , and JSTOR were included in the search to obtain all relevant, credible, rigorous, and published scientific literature. The primary reviewer (first author) performed an independent search for scientific literature, which was continuously monitored by the secondary reviewer (second author). The secondary reviewer acted as a co-analyst regarding the extracted data.EBSCOCertain keywords were utilized in the search of the above-mentioned databases: Inhibition OR Inhibitory control OR Cognitive Inhibition OR Response Inhibition OR Interreference Control AND Type 1 Diabetes OR Type 1 Diabetes Mellitus OR T1D OR T1DM OR ‘Insulin dependent Diabetes OR Juvenile Diabetes AND Self-management OR ‘Behavioral Management’ OR ‘Diabetes care plans’ AND ‘Executive functioning OR Executive Function OR Executive Dysfunction OR Cognitive Dysfunction. Studies were excluded if they focused only on T1DM, inhibition, or behavior management alone. No specified age ranges were used in the search, as studies may provide valuable information regarding differences among age groups. Furthermore, studies were included only if they adhered to ethical and methodological principles. A total of 1,068 articles were identified during the initial search. After excluding duplicates and conference proceedings, 926 remained. In the subsequent appraisal process, article titles and abstracts were reviewed in accordance with the inclusion and exclusion criteria of the study. The inclusion criteria for the study were scientific literature that focuses on (1) inhibition, (2) T1DM, and/or (3) behavior management. The exclusion criteria of the study were scientific literature that (1) focuses on only one of the above-mentioned criteria and (2) focuses on type 2 diabetes. The above-mentioned criteria were utilized when evaluating the relevance of the scientific literature identified. Based on the titles and abstracts of the scientific literature, 62 articles progressed to the next stage, in which the full text was reviewed in order to determine the relevance of the article to our research. A total of 15 remained, three of which did not meet the criteria for ethical and methodological standards. Therefore, 12 articles were included in this review. The primary and secondary reviewers consulted during the process, and consensus was reached regarding the inclusion and exclusion of articles. The thematic analysis process, as put forth in Braun and Clarke , was folOnce the second step was concluded, the third step commenced, in which the previously generated codes were utilized , and corPhases 2 through 5 did not only occur once, but continuously, and in a bidirectional manner, and this enabled the primary reviewer to continuously analyze and adjust the themes. Writing up of the findings occurred in phase 6, and the report produced was clear, concise, and non-repetitive.Two themes with related subthemes emerged from the analysis of the scientific literature identified (1) the influence of T1DM on inhibition and (2) the influence of poor inhibition on the self-management of T1DM. The findings, in general, indicate that inhibition may play a vital role for individuals living with T1DM. The results also indicate that inhibition is negatively affected by T1DM and is, therefore, associated with poorer management of the condition. The majority of the scientific literature revealed a significant relationship between T1DM, inhibition, and the behavior management of individuals with T1DM. The findings are not definitive but do indicate that there is an urgent need for future research, especially when one considers the cycle of effects that arise from the findings.The first theme that arose pertains to the effect that T1DM may have on inhibition in individuals living with T1DM. This theme is, therefore, defined as the manner in which T1DM may have an effect on inhibition. Differences occurred in the literature as to the manner in which inhibition is affected by the T1DM condition.1c alone, are related to inhibition difficulties in T1DM.” Therefore, these findings suggest that it is not merely a high level of glycemia that results in difficulties with inhibition, but the complications that arise because of hyperglycemia. Hyperglycemia may result in neuropathy (nerve damage) and microvascular complications, thereby affecting neurons within the brain ”. What was of interest in the study of Rovet and Alvarez  found that inhibition was lower in adolescents with T1DM than in control group participants, whereas Rovet and Alvarez  found loThe lack of prefrontal cortex development in children and adolescents may offer an explanation as to why early-onset diabetes can have a significant effect on their inhibition performance. One may argue that because inhibition is not yet fully developed, performance will be lower, and despite the developmental evidence that children and adolescents will not have the full capacity to utilize inhibition: when compared to control group participants, inhibition was consistently lower in children and adolescents living with T1DM than in their control group peers.One of the main subthemes that arose was that individuals living with T1DM were reported to have, in general, lowered inhibition. This theme is, therefore, simply defined as the impact of T1DM on inhibition performance, without reference to specific factors. The findings and discussion of the scientific literature failed to report what specific factor of T1DM led to lowered inhibition performance. The focus of these studies was to determine the relationship between executive functioning and T1DM. Due to inhibition being a subscale on the assessments and measurements used, authors briefly depicted the lowered inhibition scores when compared with control group participants. The results of these studies indicated that individuals with T1DM had lower inhibition scores when compared to control group participants. Despite the ambiguous nature of these results, this finding is an important factor in this critical review, as it points to the fact that aspects of T1DM contribute to lower inhibition. More importantly, it seems that an aspect of T1DM, such as glycemia, does not affect inhibition independently. It is rather multiple factors that are interacting with one another. The general impact further indicates that despite adequate control regarding T1DM, the effects on inhibition are still evident, as other factors also play a vital role . These oDuring the analysis, two studies found that T1DM may have an impact on certain structures or brain regions that are responsible for inhibition. Caruso et al.  state thWhat is of interest to note is that Rovet and Alvarez , as brie1The first theme described the effect that aspects related to T1DM, such as age at onset and fluctuating glycemia, on inhibition. The cycle that arose out of the findings is continued in theme 2, which describes the effect of poor inhibition (because of T1DM) on the behavior management of individuals living with T1DM. Inhibition, as an executive function, may play an important role in the self-management behavior of an individual living with T1DM, because of its important role in controlling thoughts, emotions, and behavior . This thThe first subtheme can be defined as the difficulty among individuals living with T1DM of inhibiting, stopping, or controlling impulsive behaviors or decisions. During the analysis, it became evident that individuals who live with T1DM often find it difficult to control impulses, as there is a significant correlation with poor control over self-management behaviors, such as control of glycemia. Broadley et al.  highlighThe study of Rovet and Alvarez  found thThe subtheme behavioral control is defined as the ability to inhibit, regulate, control, or perform certain behaviors that may influence T1DM management. Within the findings of this review, the scientific literature indicates that inhibition is strongly correlated with behavior regulation . Miller Related to adherence in T1DM are the findings of Hamburger et al , who staInhibition has also been correlated with the ability to control emotions; for example, Caruso et al.  found thThe findings of this study indicate that inhibition plays a role in the behavior management of individuals living with T1DM. These findings most importantly highlight a vicious cycle that occurs between T1DM, inhibition, and the management of T1DM. First and foremost, T1DM has an effect on inhibition and its performance. It was found that inhibition may be affected by several factors associated with T1DM, such as hypo- or hyperglycemia and the individual’s age at the onset of disease. These factors or influences affect the neurodevelopment and functioning of an individual living with T1DM, thereby lowering the ability of an individual to utilize inhibition. In turn, as seen in the second theme, poor inhibition may result in poor adherence to treatment plans for, and self-management of, T1DM. Individuals living with T1DM may find it difficult to control their behaviors, impulses, and emotions and, therefore, to undergo treatment and achieve desired health outcomes. When one considers the fact that poor management and adherence behaviors may result in fluctuating glycemia, seizures, and neurological changes, one realizes that these factors may also affect inhibition performance. The limitations of this research study relate first to the limited and scarce literature available for review and analysis. The amount of scientific literature identified was greater than expected; however, exploration and discussion of inhibition were limited. Therefore, the findings of this study cannot accurately, or with conviction, assert that a definitive relationship exists between these three constructs . Despite this, the significance of this research study remains, as it raises a need for further exploration and investigation. Furthermore, the scientific literature identified did not consider the three constructs in the context of a relationship. The limited literature thus affects the trustworthiness of the findings. Another limitation of this study is that there is no scientific literature included in a South African context. Therefore, it is difficult to translate the findings of this review to a national context. This study does, however, highlight the need for the exploration of this relationship in a South African context.Furthermore, the wide age range of participants in the articles included in the review is a strength, as it allowed for findings to arise regarding the role played by age at onset of T1DM on inhibition performance.It is recommended that future research focuses specifically on inhibition in individuals living with T1DM. It is also recommended that future research considers the relationship between T1DM, inhibition, and behavior management, as the findings of the present study indicate that such a relationship may exist. Furthermore, one of the studies included developed an intervention focusing on behavioral management and T1DM, resulting in increased inhibition. Therefore, future researchers, in a national and international context, may consider developing and exploring possible interventions that can be applied to individuals living with T1DM.T1DM is a severe and chronic condition that disrupts the lives of affected individuals and their families. Often individuals may experience hypo- or hyperglycemia as a result, both of which severely affect the functioning of the individual and may lead to negative health outcomes. The consequences of T1DM have been correlated with poor cognitive functioning and neuroanatomical changes because of the fluctuating glycemia. One of the aspects that have not been thoroughly investigated, and for which research is sparse, is inhibition. Inhibition may play a significant role in T1DM, as it plays a significant role in behavioral management. The findings of the present study indicate that inhibition does play a role in T1DM. More specifically, it was found that inhibition is affected by T1DM, and that this results in poor adherence and management of the condition. In conclusion, it is vital to consider that a vicious cycle may be present between T1DM, inhibition, and behavior management, which may in turn result in negative health outcomes in individuals with T1DM.NR was responsible for the development and execution of the research study. This included the data gathering and analysis, and writing of the manuscript. ED was responsible for the development and monitoring of the research study. This included data gathering, data analysis, and the writing and editing of the manuscript. ED provided key insights into T1DM. KB assisted in the conceptualization and development of the research study. KB played a vital role in the monitoring and editing of the manuscript, and provided key insight into executive functioning. All authors contributed to the article and approved the submitted version.


Ancylonema nordenskiöldii and Ancylonema alaskanum  reduce the bare ice albedo of the Greenland Ice Sheet, amplifying melt from the largest cryospheric contributor to eustatic sea-level rise. Little information is available about glacier ice algae interactions with other microbial communities within the surface ice environment, including fungi, which may be important for sustaining algal bloom development. To address this substantial knowledge gap and investigate the nature of algal-fungal interactions, an ex situ co-cultivation experiment with two species of fungi, recently isolated from the surface of the Greenland Ice Sheet , and Articulospora sp.), and the mixed microbial community dominated by glacier ice algae was performed. The utilization of the dark pigment purpurogallin carboxylic acid-6-O-β-D-glucopyranoside (C18H18O12) by the two fungi was also evaluated in a separate experiment. P. anthracinoglaciei was capable of utilizing and converting the pigment to purpurogallin carboxylic acid, possibly using the sugar moiety as a nutrient source. Furthermore, after 3 weeks of incubation in the presence of P. anthracinoglaciei, a significantly slower decline in the maximum quantum efficiency  in glacier ice algae, compared to other treatments, was evident, suggesting a positive relationship between these species. Articulospora sp. did uptake the glycosylated purpurogallin, but did not seem to be involved in its conversion to aglycone derivative. At the end of the incubation experiments and, in conjunction with increased algal mortality, we detected a substantially increasing presence of the zoosporic fungi Chytridiomycota suggesting an important role for them as decomposers or parasites of glacier ice algae.Heavily pigmented glacier ice algae The online version contains supplementary material available at 10.1007/s00248-022-02033-5. Ancylonema alaskanum ) in relative units as rETR = Y(PSII) × PAR × 0.5, assuming an equal division of PAR between photosystems I and II. Analysis of RLCs (rETR ~ PAR) followed Perkins et al. (2006), with iterative curve fitting  and calculation of the relative maximum electron transport rate (rETRmax), maximum light utilization coefficient (α) and light saturation coefficient (Ek) following Eilers and Peeters , psychrotolerant. Optimum growth temperature: 15–24 °C. CYA/CYAS: 1.1 [0.8–1.4], halotolerant. Conidia en masse dark green.Synnemata or fasciculation: none; sclerotia and ascomata: none; colony texture on CYA: velutinous, radially furrowed; conidium colour on CYA: green to dark green; exudate droplets on CYA: none; reverse colour on CYA: pale yellow; reverse colour on YES: cream yellow; diffusible colour: none; Ehrlich reaction: no or weak violet reaction; conidiophores arising from agar surface; stipes 100–)150–300(–500) × 3–4 (6.5) μm, smooth-walled; penicilli predominantly terverticillate; rami 2–3 per stipe, mostly appressed, (7–)8.5–20 × 2.5–5.5 (–8) μm; metulae 3–4 per ramus, 7–13 (–17) × 3–6 μm, clavate to apically inflated up to 10 μm diam; phialides 4–6 per metula, ampulliform with distinguishable necks, 5.5–11 × 2–4 μm; conidia subglobose to ellipsoidal, 2.5–3.5 × 2–3 μm, walls finely roughened, born in chains that form loose masses , refrigerators.Chemotaxonomy  at the Department of Biology, Biotechnical Faculty, University of Ljubljana, Slovenia. Accession numbers of DNA sequences derived from ex-type strain EXF-11443, deposited into the above mentioned Ex Culture Collection: MT080493 (BenA), MT080519 (RPB2), MT080552 (CaM).The holotype, EXF-11443H, originated from dark ice in Greenland Ice Sheet, approximately 35 km from the southwestern Ice Sheet margin, 67° 04′43″ N 49° 20′29″ W, adjacent to the S6 PROMICE weather station, in July 2017 by Laura Perini. It is permanently preserved in a metabolically inactive state at the Ex Culture Collection of the Infrastructural Centre Mycosmo (MRIC UL), Slovenia  was detectable over RLCs performed using PAM fluorescence  , possibly due to the lack of light stress compared to the light treatment. Furthermore, Fv/Fm was significantly higher in glacier ice algae + Penicillium treatment in the light as compared to the algal control . On the contrary, Fv/Fm was not significant for the glacier ice algae + Articulospora treatment as compared to the algal control in the light  or the dark . No significant differences in α or Ek were apparent between treatments of light/dark incubations , with nts Fig. . To giveradiance . Values radiance . After 5nce Fig. . Considence Fig. , likely ons Fig.  despite −1) Fig. . Fv/Fm uons Fig.  and S7.FArticulospora sp. had densely septated brownish hyphae with many vacuolar inclusions (not shown). No sporulation of Articulospora sp. during the experimental incubation was observed. During its growth in co-culture with algae, it formed a net-like hyphal structure that incorporated individual algal cells . Both fungi grew in an extremely low nutrient medium (melted ice) and at a low temperature (+4 °C).Microscopy showed that after 3 weeks of incubation, glacier ice algae remained pigmented and formed clumps containing also mineral particles Fig. . Articullls Fig. . P. anthmps Fig. . After 2P. anthracinoglaciei treatment under dark conditions, where glacier ice algae retained their characteristic pigmentation  showed in solution (extracellularly) the presence of 337.5 ±162.5 mAU of purpurogallin glucopyranoside and a lower amount of the aglycone derivative (1.1 ±0.9 mAU)  than purpurogallin glucopyranoside peak (31.25 ±1.75 mAU), which represented a 157 ±9% interconversion within the Penicillium treatment. Articulospora sp. mycelium contained 46 ±2 mAU of purpurogallin glucopyranoside and 1 ±0 mAU of purpurogallin aglycone, with a lower percentage of interconversion (2.2 ±0.1%).The uptake of purpurogallin glucopyranoside by AU) Fig. . Fungi aIn this study, we have attempted to map interactions between glacier ice algae  and fungal communities isolated from the surface of the GrIS. Whilst fungal concentrations were most likely elevated as compared to ambient concentrations apparent within GrIS surface ice, the purpose of our experiments was not to recreate ambient concentration, but to promote a response between the partners. Fungal concentrations were therefore applied as a treatment to identify the fungal impact on glacier algal assemblages. The microbial community in glacial environments is complex and the interactions between microorganisms may well be more elaborate than implied here. Particularly, besides filamentous fungi that were tested here, abundant species of yeast might have a role in modulating the growth and survival of ice algae.P. anthracinoglaciei improving the photosynthetic capabilities of the ice algae, compared to the other treatments. The two fungal species used in the experiments also produced several active compounds, emphasising their ability to regulate the growth of other microbial partners in these associations  in co-cultures that contained P. anthracinoglaciei in comparison with other treatments, indicating that Penicillium improved the survivability of glacier ice algae. Whilst this might simply be a result of a shading effect conferred by the fungi, no such positive effect on Fv/Fm was conferred by Articulospora sp. that entrapped algal cells in its hyphal network and would presumably have the same shading impact on glacier ice algae. The beneficial effects of P. anthracinoglaciei on algae in vitro might be driven additionally by the production of beneficial secondary metabolites, or mobilization of otherwise unavailable organic or inorganic compounds. For example, several species of penicillia are able to mobilize phosphorus, which has been demonstrated to be a limiting nutrient for glacier ice algae communities [P. anthracinoglaciei showed its ability to produce several secondary metabolites in standard conditions   Table . These mrganisms . Xanthoecissimum . Breviona et al. , are bioia Table , Fig. S2P. anthracinoglaciei on glacier ice algae pigmentation, as evidenced by the prevalence of fully pigmented  glacier algal cells (73%) following 5 months of incubation in the dark  can involve the presence of purpurogallin glucopyranoside in the extracellular environment, these data do not contrast with the ability of Penicillium to sustain the algal pigmentation after a long period of incubation in the dark. The fungal ability to utilize the glucopyranoside sugar as a nutrient source will be confirmed in a future experiment.Incubation of ime Fig. . These dArticulospora sp. exhibited a potential lichenoid-like relationship with glacier ice algae. Both light and scanning electron microscopies showed a progressive accumulation of the glacier ice algae, embedded in extracellular polymeric substances (EPS), in Articulospora hyphal network, that became particularly distinct after 2 months of incubation. Articulospora sp. showed the ability to uptake purpurogallin glucopyranoside in the mycelium; however, its interconversion into purpurogallin aglycone was negligible compared to Penicillium in pure culture with purpurogallin glucopyranoside in the medium.Articulospora sp. did not highlight any significant increase in the glacier ice algae Fv/Fm , either in light or dark treatments, suggesting a neutral effect  on the algal photophysiology, and/or implying that the incubation conditions may not have been optimal to test the interactions between glacier ice algae and Articulospora.According to the literature, in borderline lichens, two participating species do not form structures resembling a lichen thallus. In these associations, fungal and algal cells grow intertwined with loose contacts observed in both native samples and culture experiments , 61. BecA. nordenskioeldii were found to be colonized by chytridiomycetous fungi in ice samples from Svalbard glaciers [Although this study was not initially designed to investigate the role of Chytridiomycota, a considerable growth of this fungal group was observed by light microscopy in conjunction with increased algal mortality at the termination of our microcosm experiments (after 5 months). Chytridiomycota were only observed sporadically and in smaller numbers during previous sampling time points of the experiment. However, the presence of unidentified Chytridiomycota in dark ice samples from the GrIS was also supported by NGS biodiversity analysis , confirmglaciers , hintingglaciers , the losglaciers . No infoglaciers .A study of interactions between glacier ice algae communities and two selected fungal species found in the supraglacial ice of the Greenland Ice Sheet indicated that fungi and algae have different types of interactions.P. anthracinoglaciei suggests its ability of metabolizing the sugar moiety of the pigment, possibly using it as a carbon source. Conversely, Articulospora sp. embedded algae into its hyphal network, but failed to provide the algae with any benefits based on the analyses conducted in the experiments. Given the complexity of the systems used in this study, investigation of the potential involvement of additional partners in the fungal-algal interactions would also shed more light on the impact of non-photosynthetic organisms on the GrIS algal blooms.The interconversion of the pigment purpurogallin glucopyranoside into its aglycone derivative by Penicillium anthracinoglaciei and Articulospora sp.) could have a role in sustaining glacier algal blooms during the ablation season or during the over-wintering period, providing various benefits from the mobilization of organic or inorganic compounds, to the secretion of secondary metabolites, physical shielding from light or water currents, induction of dormancy or even modulation of the growth of other microorganisms in putative lichenoid-like structures.On the one hand, the increasing presence of Chytridiomycota at the end of the incubation experiment in parallel with higher algal mortality indicates that these zoosporic fungi could act as decomposers or parasites of algal blooms. On the other hand, other fungal species Below is the link to the electronic supplementary material.


The helical arrangement of cardiac muscle fibres underpins the contractile properties of the heart chamber. Across the heart wall, the helical angle of the aligned fibres changes gradually across the range of 90–180°. It is essential to recreate this structural hierarchy in vitro for developing functional artificial tissue. Ice templating can achieve single-oriented pore alignment via unidirectional ice solidification with a flat base mould design. We hypothesise that the orientation of aligned pores can be controlled simply via base topography, and we propose a scalable base design to recapitulate the transmural fibre orientation. We have utilised finite element simulations for rapid testing of base designs, followed by experimental confirmation of the Bouligand-like orientation. X-ray microtomography of experimental samples showed a gradual shift of 106 ± 10°, with the flexibility to tailor pore size and spatial helical angle distribution for personalised medicine. The muscle fibre orientation across the heart wall is a highly complex three-dimensional 3D) arrangement, which constantly changes throughout the cardiac cycle (diastole and systole). The muscle fibres form layers that wrap helically around the heart chamber, and the helix angles in the layers vary gradually across the heart wall. As shown in D arrangeFor artificial cardiac constructs, replicating such 3D architectures in tissue engineering scaffolds remains a problem. Local alignment on the cellular level has been achieved using a variety of methods, including electrospinning ,10,11, fOne of the benefits of ice templating is its simplicity and the potential to fabricate architectures with interconnected pore networks, which is pivotal to 3D cell culture. Cells can recognise anisotropic pore structures and show cytoskeletal alignment with the orientation of pore walls ,27; hencHere, we describe how we successfully recreated the complex helical architectures across the heart wall in ice-templated collagen scaffolds via multi-directional freezing. We utilised finite element modelling as a rapid design tool to tailor the 3D thermal gradient to induce ice growth to match the helical angle variation across the heart wall. We then propose an experimental design with a 5-wedge base, as shown in ®. Two mould designs were investigated to study the influence of base geometry: the wedge base design and the Bouligand-like orientation design. Both moulds were constructed using a copper base and a polycarbonate body, and the freezing of the slurry was simulated by water/ice phase change. The heat transfer during the freezing stage of ice-templating was simulated in COMSOL Multiphysics 5.6The phase change between water (w) and ice (i) was simulated using a transitional mushy zone, which was defined as a temperature zone over a 4 °C interval centred at 0 °C. The phase proportion –(5), where 2K) for all simulations [Three boundary conditions were imposed during the simulations: the initial temperature, heat extraction at the mould bottom, and heat exchange with the surrounding environment. The initial temperature was set to be 20 °C for all domains. The heat extraction at the mould bottom was set to be in line with a freezing protocol. For both the wedge base mould and the Bouligand-like orientation mould designs, the temperature of the bottom surface of the base (the contacting surface between the mould and the cold finger) was set as −10 °C for 1 min of stabilisation at the start of the simulation, followed by continuous cooling at the rate of 1 °C/min. Finally, the heat exchange with the surrounding environment was simulated using convective heat flux. Equation (6) was used to calculate the heat flux, where ulations ,43.(6)q1 wt% insoluble bovine dermal collagen powder was hydrated in 0.05 M acetic acid overnight. The collagen suspension was then blended and degassed under vacuum to form a homogeneous slurry. The slurry was then poured into the mould under study. The mould containing the slurry was placed on a copper finger of lab-built freezing equipment . This cuy-axis were evenly divided into 5 substacks, and then the directionality analysis was performed on individual slices. The 3D orientation distribution in certain regions combined all slices from one substack. The directionality histogram was then normalised to a maximum intensity of 1 for comparison.The scaffolds that were freeze-dried using the prototype mould were cut into a prism volume of interest, as suggested in N = 4 for the samples freeze-dried using the prototype mould; N = 3 for the samples freeze-dried using the refined mould for the two freezing protocols. The error shown represents the standard deviation unless otherwise stated in the text.® to investigate the effects of the slope angle . Every 100 s, linear regression was implemented to evaluate the IFF slope variation as simulations progressed. It was found that the IFF angle initially followed the base angle and decreased over time as the IFF moved towards the top. It was also noted that as IFF was near the top, the linear regression became less reliable due to the mould edges. To further quantify the influences of the base geometry, we used the position of the ice freezing front (IFF) at every 100 s interval throughout the simulation process. The IFF was represented by the interface of −2 °C to avoid complications of the latent heat, and this separated the domain into two phases, as suggested in x-axis (denoted as z-axis), denoted as y-axis exhibited a 3D mirror symmetry with respect to the x-z plane in the centre of the prism. Above one individual base, the 3D streamlines were flattened onto a 2D plane and orientation analysis was performed . It was also possible to obtain different orientation shifts within the same ice-templated scaffold. Let the region at the centre of the prism be at the reference height as 0 along the In this work, we have explored the 3D directional freezing with the aid of finite element simulations and proposed a creative solution to replicate the helical arrangement of the cardiomyofibres across the heart wall. We started with a simple wedge base mould design. The simulation results showed clear evidence that the slope angle affected the pore growth orientation, especially near the base. It was noted that the high thermal conductivity of the copper base allowed fast, almost simultaneous ice nucleation at the very surface of the base, and the geometry of the base had little effect on delaying the temperature from reaching the nucleation temperature across the entire base. Hence, a thin isotropic layer near the bottom was expected but considered negligible. Then, the ice growth was influenced by both the thermodynamics of crystal growth and the presence of mould boundaries. In this case, the combined effects led to a 2D thermal gradient that changed the initial pore orientation from the vertical direction to the direction perpendicular to the wedge base. This perspective was overseen in the previously reported work due to the choice of base materials. If the wedge was made of a poor thermal conductor, such as PDMS, then the ice nucleation would be delayed because of the horizontal thermal gradient. The combined effects would eventually lead to a lamellar structure, as demonstrated in the work of Bai et al. and Zhang et al. ,38. On tThen, we proposed a base design comprising five wedges to create a 3D thermal gradient with mirror symmetry that resulted in a Bouligand-like pore orientation. Similar to the wedge base design, we expected fast ice nucleation at the base surface, followed by ice growth, whose direction was heavily influenced by the thermal gradient. Both the simulation and the pilot study with the prototype mould suggested a successful pore orientation shift, confirming our hypothesis that the 3D pore orientation distribution can be controlled by the base topography via a thermal gradient. The experimental sample showed a pore orientation variation of 106 ± 10°, within the 90–180° range of the transmural helical angle change in vivo ,3,4. OurFinally, we developed a refined mould to adjust the dimension of the ice-templated collagen scaffolds to clinically relevant sizes  with tuneable architectural features, such as pore size, heart wall thickness, and alignment direction distribution. Our previous work has shown that it is possible to fine-tune the pore size via freezing protocol  and to aIn summary, we established a robust platform based on predictive modelling and experimental confirmation, successfully recreating the structural hierarchy of the native heart in ice-templated collagen scaffolds. Our computationally designed scaffolds offer a simple, scalable, and adaptable tool to build artificial constructs with customised 3D distributions of pore orientation.