The nasal cannula is considered a trusted and effective means of administering low-flow oxygen and is widely used for neonates and infants requiring oxygen therapy, despite an understanding that oxygen concentrations delivered to patients are variable.2) through nasal cannulas during low-flow oxygen delivery. Parameters influencing variability in FiO2 were evaluated, as was the hypothesis that measured FiO2 values could be predicted using a simple, flow-weighted calculation that assumes ideal mixing of oxygen with entrained room air. Tidal breathing through neonatal and infant nasal airway replicas was controlled using a lung simulator. Parameters for nasal cannula oxygen flow rate, nasal airway geometry, tidal volume, respiratory rate, inhalation/exhalation, or I:E ratio (ti/te), breath waveform, and cannula prong insertion position were varied to determine their effect on measured FiO2. In total, FiO2 was measured for 384 different parameter combinations, with each combination repeated in triplicate. Analysis of variance (ANOVA) was used to assess the influence of parameters on measured FiO2.In the present study, realistic nasal airway replicas derived from medical scans of children less than 3 months old were used to measure the fraction of oxygen inhaled (FiO2 was not appreciably affected by the breath waveform shape, the replica geometry, or the cannula position but was significantly influenced by the tidal volume, the inhalation time, and the nasal cannula flow rate.Measured FiO2 for measured values above 60%, but an empirical correction to the calculation provided good agreement with measured FiO2 across the full range of experimental data.The flow-weighted calculation overpredicted FiO Oxygen therapy is used globally to treat hypoxemia in young children with a wide range of respiratory conditions, including pneumonia, bronchiolitis, and acute asthma. Administration of low-flow oxygen through nasal cannulas is the preferred delivery method for children under five years of age who require oxygen therapy . For neo2) delivered to neonates and infants is not straightforward. Variability in FiO2 with these devices is likely due, at least in part, to variation in breathing pattern, which influences the entrainment of room air that mixes with supplied oxygen within the upper airway. In the absence of established methods for estimating FiO2, clinicians and researchers have extrapolated from studies in adults [2 as indeterminable [2 delivered to young and vulnerable patients has the potential to adversely influence clinical outcomes, given that the consequences of supplying too low, or too high, an oxygen concentration can be severe [For open patient interfaces such as nasal cannulas, estimating the fraction of inspired oxygen , tidal volume (Vt), and the shape of the breathing waveform. Two breathing waveforms were initially examined to evaluate whether the waveform shape significantly influence measured values of FiO2. These preliminary measurements were made using two of the seven replicas (subjects 4 and 8). Commonly, prior studies have employed sinusoidal waveforms as an idealized functional form. Here, in addition to a sinusoidal waveform, a triangular waveform, which approximates clinically-observed waveforms for infants with chronic lung disease, was investigated [Simulation of tidal breathing required the following parameters to be specified: respiratory rate (RR), I:E ratio .Figure For collection and synchronization, the oxygen concentration-versus time data from the analyzer and the volume-versus-time data from the lung simulator were streamed through an RS-232 serial port and TCP/IP connection, respectively, to a LabVIEW virtual instrument . Volume-versus-time was numerically differentiated to calculate flow rate-versus-time. In each experiment, the flow of oxygen to the nasal cannula was initiated prior to starting the lung simulator in order to produce a characteristic sudden drop in oxygen concentration at the start of the first simulated breath. This signal was used to synchronize concentration and flow-rate data in time. After synchronization, oxygen concentration and flow waveforms were observed to be consistent with expected characteristics [2 was then determined as the ratio between the volume of inhaled oxygen and the tidal volume. For each experiment, FiO2 was averaged over five consecutive breaths after a steady state in expiratory oxygen concentration was observed (after at least 50 breaths). The experiment was repeated in triplicate for each combination of breathing parameters, cannula insertion position, and airway replica.The volume of oxygen inhaled past the trachea during each breath was determined by integrating the product of oxygen concentration and flow rate using the trapezoid rule. Similarly, the inhaled tidal volume was calculated by integrating the flow rate over each inhalation cycle. The FiO2 in the mixed inspiratory flow was estimated using a flow-weighted calculation that assumes ideal mixing of oxygen with entrained room air:During low-flow oxygen delivery through nasal cannulas, the oxygen concentration in gas inhaled past the upper airway is determined by the mixing of oxygen supplied through the cannula with the entrained room air that forms the bulk of the inhaled breath. As with previous studies evaluating oxygen delivery for infants  and adult is the inhaled tidal volume, ti is the inhalation time, Qi is the average inhalation flow rate, QNC is the nasal cannula flow rate, and the room air oxygen concentration is assumed to be 21%.Here V2 calculated using Eq. , and nasal cannula position . Results with a two-sided P value less than 0.05 were considered significant.ANOVA procedures employing a set of one-factor repeating measures with Tukey post hoc analysis were used to assess the influence of each parameter on FiOc, developed by Lawrence and Lin [2 values. This coefficient ranges between 0 and 1, with a higher value indicating better agreement between predicted and measured values.In addition, the concordance correlation coefficient, ρ and Lin , was use2 (P = 0.48). Accordingly, all further experiments were conducted using sinusoidal waveforms.Preliminary measurements conducted in two replicas (subjects 4 and 8) across the full range of breathing parameters described above revealed no significant influence of the sinusoidal versus triangular waveform on measured FiO2 averaged across the replica geometries with cannula prongs fully inserted into the nares for each combination of tidal volume, respiratory rate, I:E ratio, and nasal cannula flow rate. Across the full parameter range, neither the replica geometry (P = 0.97) nor the cannula position (P = 0.88) influenced measured FiO2. Tabulated values of FiO2 were instead influenced by the tidal volume, respiratory rate, and nasal cannula flow rate (P < 0.05). At the two levels evaluated, the I:E ratio did not independently influence FiO2 (P = 0.42). However, the I:E ratio and the respiratory rate together determined the inhalation time, which influenced FiO2 (P = 0.001), as expected from Eqs. 2.Table  P = 0.00, as expe2 values predicted using Eqs.  is introduced. Overall, the use of Eq.  influenced measured FiO2.The present study evaluated FiO2 delivered through nasal cannulas to neonates and infants, even at fixed nasal cannula flow rates [2 was determined by variation in two breathing parameters: tidal volume and inhalation time. Monitoring, or estimating, these parameters is therefore key to improving estimation of FiO2 in future research or clinical practice. Accurate estimation of FiO2 would be useful when comparing new modes of respiratory support to standard, low-flow oxygen therapy [2/FiO2 or SpO2/FiO2 ratios for patients receiving low-flow oxygen delivered via nasal cannula [2 estimation may help to avoid any deleterious consequences of supplying FiO2 outside of safe, target ranges [These results help to explain previous observations of high variability in FiOow rates , 11, 12. therapy ; it migh cannula , 5. Moret ranges , 7.. [2 may be estimated using Eqs. . The authors attributed differences between predicted and measured values to their use of assumed, rather than measured, values of tidal volume and inhalation time in making their predictions. Benaron and Benitz [Based on data obtained using an in vitro methodology like that used in the present study, but with airway replicas and simulated breathing patterns representing adult subjects, Katz et al.  previousing Eqs. –3, whichz et al. , who alsz et al. , althougz et al.  presenten et al. . Howeverd Benitz  also notd Benitz  ranged id Benitz , 38, neid Benitz .2 values below approximately 60%  of 0.5. Above this value, as the flow rate of oxygen supplied through the nasal cannula approaches the average inhalation flow rate, it is likely that a fraction of supplied oxygen is lost to the room environment during periods of inhalation where the nasal cannula flow rate exceeds the instantaneous inhalation flow rate. Such losses are not accounted for in Eqs. , the omission of gas exchange is expected to bias measured FiO2 upwards on average by 1.6% (absolute) at the lowest tidal volume studied (17.1 ml). At the highest tidal volume studied, 36.6 ml, this upwards bias is reduced to 0.7%. In either case, the change in measured FiO2 owing to gas exchange is smaller than the precision of the repeated experiments and not anticipated to be clinically important.A limitation of the present study is the use of rigid airway replicas and a lung simulator as opposed to conducting measurements in vivo in human neonates and infants. While use of an in vitro approach permitted controlled and repeatable experiments that would be extremely challenging to conduct in vivo in this age group, this advantage comes with trade-offs. Notably, rigid airway replicas do not deform dynamically with breathing flow rate during the inhalation-exhalation cycle or with administration of small amounts of positive airway pressure. However, the potential influence of dynamic variation in nasal airway geometry on the present results is expected to be insignificant. Such variation is small compared with the intersubject variation in geometry across the seven district airway replicas used in the present study, where replica geometry was found not to significantly influence measured FiOmely low , gases c in vivo . In the 2 delivered to neonates and infants during low-flow oxygen therapy administered through nasal cannulas. The flow rate of supplied oxygen, in combination with tidal volume and inhalation time, was found to determine FiO2. Neither the nasal airway geometry, nor the position of cannula prongs in the nares  influenced measured FiO2. An empirical correlation was presented that provides good agreement with measured FiO2 across the full range of experimental data, from 27.9 to 88.6%. Improved prediction of FiO2 delivered to neonates and infants receiving standard, low-flow oxygen therapy is expected to be of value in maintaining oxygenation within safe, target ranges, and may contribute to increased use of risk prediction ratios involving FiO2.Realistic nasal airway replicas and simulated breathing patterns were used to evaluate the FiO


Qibin Tongbian decoction in the treatment of diabetic constipation and its influence on the intestinal environment and the incidence of adverse reactions.  This study aimed to study the clinical efficacy of Qibin Tongbian decoction . Outcome measures included clinical efficacy, intestinal environment, and incidence of adverse reactions.  In this prospective study, a total of 62 patients with diabetic constipation admitted to our hospital from June 2020 to March 2021 were recruited and assigned at a ratio of 1 : 1 to receive either traditional treatment (control group) or P < 0.05). Qibin Tongbian decoction resulted in significantly lower fasting plasma glucose (FPG) levels and 2-hour postprandial plasma glucose (2hPG) levels versus conventional treatment (P < 0.05). Patients receiving Qibin Tongbian decoction showed significantly lower constipation symptom scores and PAC-QOL scores versus conventional treatment (P < 0.05). Qibin Tongbian decoction resulted in a significantly larger number of bifidobacteria and enterococci and a smaller number of yeasts versus conventional treatment (P < 0.05). Qibin Tongbian decoction was associated with a significantly lower incidence of adverse reactions and recurrence rate versus conventional treatment (P < 0.05).  Qibin Tongbian decoction was associated with significantly higher efficacy and a lower TCM symptom score versus conventional treatment (Qibin Tongbian decoction combined with conventional TCM therapy yields a promising effect in the treatment of diabetic constipation. It can improve the intestinal environment of patients, mitigate the symptoms of constipation, enhance the quality of life of patients, and reduce the incidence of adverse reactions, so it is worthy of promotion and application. Qibin Tongbian decoction combined with conventional TCM treatment of diabetic constipation, and its influence on the intestinal environment and the incidence of adverse reactions in patients.As a common clinical symptom of diabetic autonomic neuropathy involving the digestive system, diabetic constipation is associated with the damage of intestinal mucosal epithelial cells caused by increased blood sugar, decreased sensitivity of the large intestine, and autonomic neuropathy of the large intestine , 2. ExceP > 0.05). The study was approved by the Ethics Committee of Nanjing University of Chinese Medicine with ethics certificate number 2019-12-11, and all patients and families gave their informed consent.In this prospective study, a total of 62 patients with diabetic constipation admitted to our hospital from June 2020 to March 2021 were recruited and assigned (1 : 1) to a control group or an experimental group. The control group consisted of 13 males and 18 females, aged 48–69 (55.44 ± 12.30) years; the course of diabetes was 4–10 (6.52 ± 3.81) years; and constipation course was 1–4 (2.61 ± 1.56) years. The experimental group consisted of 15 males and 16 females, aged 45–70 (56.53 ± 11.96) years; the course of diabetes was 3–9 (5.87 ± 3.74) years; and constipation course was 2–5 (3.22 ± 1.69) years. The two groups showed similar baseline data , 5 mg each time, 3 times a day, for 2 weeks. The experimental group received Guiding Principles for Clinical Research of New Chinese Medicines [(1) TCM symptom score and clinical efficacy: the TCM symptom score of the two groups of patients was assessed with reference to the edicines . The clin(%)), and measurement data were examined by t-test and expressed as (x ± s). Differences were considered statistically significant at a P value of <0.05.The data were statistically analyzed by SPSS 20.0, and GraphPad Prism 7  was used to plot graphics. The count data were tested by the chi-square test and expressed as (P > 0.05). Qibin Tongbian decoction was associated with a significantly higher efficacy (96.67% vs. 83.33%) and a lower TCM symptom score versus conventional treatment (P < 0.05). See Tables Before treatment, the TCM symptom scores were comparable between the two groups (P > 0.05). Qibin Tongbian decoction resulted in significantly lower FPG and 2hPG levels versus conventional treatment (P < 0.05). See Figures Before treatment, the FPG and 2hPG levels of the two groups of patients were not statistically different (P > 0.05). Patients receiving Qibin Tongbian decoction showed significantly lower constipation symptom scores and PAC-QOL scores versus conventional treatment (P < 0.05). See Figures Before treatment, no statistical difference in the scores of constipation symptoms and PAC-QOL scores was observed between the two groups (P > 0.05). Qibin Tongbian decoction resulted in a significantly larger number of bifidobacteria and enterococci and a smaller number of yeasts versus conventional treatment (P < 0.05). See The two groups showed no significant differences in terms of the number of bifidobacteria, enterococci, and yeasts before treatment (Qibin Tongbian decoction was associated with a significantly lower incidence of adverse reactions (1.67%) versus conventional treatment (11.67%) (P < 0.05). See Qibin Tongbian decoction showed a significantly lower recurrence rate versus conventional treatment (P < 0.05). See There were 50 patients with effective treatment in the control group and 58 patients in the experimental group. The patients given Qibin Tongbian decoction can nourish the lung and spleen; angelica, hemp seed, white honey, and cistanche play a role in nourishing blood, moistening the intestines, and relaxing the bowels; tangerine peel exerts the effects of regulating Qi, promoting spleen, drying dampness, and reducing phlegm; white Atractylodes rhizome and Fructus aurantii have the effect of invigorating the spleen and regulating Qi; betel nut slices yield a satisfying result in regulating Qi and dredging stagnation, unblocking the accumulation; peach kernels function well in promoting blood circulation, removing blood stasis, and moisturizing the intestines; fried raspberry seeds have the property of treating constipation; and licorice can reconcile the above medicines [Diabetic constipation is caused by multiple factors, such as decreased gastrointestinal motility caused by autonomic nerve dysfunction consequent to diabetes, gastrointestinal hormone secretion disorders resulting from prolonged hyperglycemia, and imbalanced intestinal flora , 11. In edicines , 17, 18.Qibin Tongbian decoction was associated with a significantly higher efficacy (96.67% vs. 83.33%) and lower FPG and 2hPG levels versus conventional treatment, indicating that Qibin Tongbian decoction produces a favorable effect on diabetic constipation. Remarkably, patients receiving Qibin Tongbian decoction showed significantly lower constipation symptom scores and PAC-QOL scores versus conventional treatment, which was consistent with the research results of Li et al. [Qibin Tongbian decoction can mitigate the symptoms of constipation and improve the quality of life of patients. Additionally, Qibin Tongbian decoction resulted in a significantly larger number of bifidobacteria and enterococci and a smaller number of yeasts versus conventional treatment, indicating that Qibin Tongbian decoction can improve the intestinal environment of patients with constipation and boost the function of intestinal peristalsis. Furthermore, the incidence of adverse reactions in the experimental group was 1.67% and that in the control group was 11.67%, indicating that Qibin Tongbian decoction for diabetic constipation can reduce adverse reactions. Encouragingly, the patients with robust effects were followed up for 6 months, and the results showed that the recurrence rate of patients in the control group was higher than the experimental group (26% vs 6.90%), and the results of this study are in line with a previous study [Qibin Tongbian decoction can notably reduce the recurrence rate of the experimental group in contrast with the control group (16.59% vs 44.15%). The above results suggest a promising effect of Qibin Tongbian decoction in treating diabetic constipation and reducing the recurrence rate of constipation. Qibin Tongbian decoction is mostly used to nourish the kidney and strengthen the spleen. Constipation is closely related to the kidney. A deficiency in kidney Yin results in a loss of intestinal moistening and dryness, while a deficiency in kidney Yang results in an inability to warm and moisten the intestines. The spleen and stomach are the sources of Qi and blood, with the main function of transformation of nutrients, so spleen Qi deficiency will reduce the function of nutrient intake. The lung and large intestines are closely associated. Lung Qi deficiency leads to weakness of large intestine conduction, and blood deficiency prevents fluid from nourishing the large intestine; Yin deficiency of the lung results in dryness of the large intestine and loss of intestinal moistening, both of which can lead to dry stools and difficult discharge, resulting in constipation. Due to prolonged dryness, liver depression, and Qi stagnation, blood stasis blocks the ligaments, and fluid cannot penetrate the large intestine, resulting in fluid deficiency and intestinal dryness, which can aggravate the condition.In this study, i et al. , suggestus study  stating Qirong Runchang oral solution is effective in the treatment of diabetic constipation in the elderly, especially in elderly diabetic patients with deficiency of Qi and Yin, deficiency of spleen and kidney, and loss of bowel moistening as identified by traditional Chinese medicine, with a fast onset of action and a significant laxative effect. Its short-term use can significantly improve the patient's bowel movement and relieve constipation symptoms, and long-term use can ameliorate the symptoms of general body weakness, thus enhancing the patient's quality of life significantly. The drug features gentle action, long maintenance, no significant effect on glycemic control in elderly diabetic patients, and no significant adverse drug reactions, which is worthy of clinical reference and use.The innovation of this study is the use of Qibin Tongbian decoction in combination with conventional Chinese medicine for the treatment of diabetic constipation. The limitation of this study is the absence of long-term extensive follow-up data to determine the long-term efficacy of diabetic patients, which requires additional follow-up time in the future to obtain more clinical information.Qibin Tongbian decoction is a preferred option for diabetic constipation. It can improve the patient's intestinal environment and patient's constipation symptoms, upgrade the patient's quality of life, and reduce the incidence of adverse reactions, with a high safety profile.A combination of


Endangered cranes migrate to the Demilitarized Zone in Korea in search for habitat and food during winter. While cranes have the potential to influence soil biogeochemical processes via dropping, foraging, and walking, few studies have investigated ecological roles of migrating birds in the new habitat. Here, we explored how cranes alter resource landscape  and microbial community in soil. We set up control  and treatment (free crane activities) plots  in a rice paddy, and collected soils at 0–15 cm three months after the crane migration. Soils were tested for total carbon, total nitrogen, water extractable organic carbon, and Diffuse Reflectance Infrared Fourier Transform Spectroscopy, along with microbial parameters . The wintering crane activity significantly increased total carbon and nitrogen contents, but decreased the ratio of CH  to COO (carboxylic) in soil. Also, both microbial biomass and respiration was greater in soils under crane activities. Bacterial and fungal community composition differed with or without crane activities, with treatment soils harboring more diverse microbial communities. Our results demonstrate that crane migration created a distinct system with altered resource landscape and microbial community, highlighting beneficial effects of migratory cranes on the soil biogeochemical processes in rice paddies. This study may help encourage more farmers, local governments, and the public to participate in crane conservation campaigns targeted at rice fields. Habitat loss, excessive hunting, and poisoning have decreased the number of cranes, leading to 11 out of 15 crane species threatened with extinction under the International Union for Conservation of Nature (IUCN) Red List Categories system , cranes 15δN, Kameda et al. [Phalacrocorax carbo) transports N from fresh water to forest ecosystems via droppings and collection of N-rich nest material. Alternatively, birds may refresh the quality of resources via digestion or foraging. Bird fecal matter is partly or completed digested, so it may provide readily available resources to other organisms [Cranes may alter biogeochemical processes of the new habitat by recharging, redistributing, and refreshing resources during winter. Bird fecal matter is rich in carbon (C), nitrogen (N), phosphorus, and potassium –10, likea et al.  found thrganisms .Changes in the resource quantity and quality often alter soil microbial activity and community composition, with no clear directional changes –16. A meGrus japonensis) and White-naped cranes (Grus vipio) and 5 other crane species come to Cheorwon in mid-October, and leave early March for north-eastern China and Russia for breeding [Our study site is Cheorwon, Kangwon Province, South Korea. Red-crowned cranes . The mean annual temperature is 10.2°C, with a mean daily maximum of 16.2°C and mean daily minimum of 4.7°C. The mean annual precipitation is 1391.2 mm . Parent Cranes make use of the northern part of Cheorwon, approximately 10,000 ha including the rice fields in and around the CCZ, Hantan River, and the wetlands inside of the DMZ . A total-1 dry soil), respectively. Along with soil samples, crane droppings were collected from the treatment plots. All samples were kept in a cooler, sent to Korea Advanced Institute of Science and Technology, and stored at 4°C before chemical and biological analyses.The farmers allowed us to use one rice paddy among the flooded ones for this study . According to the farmers, the majority of the cranes roost in the flooded rice fields at the early phase of their migration until mid-Dec when the wetlands inside of the DMZ are frozen and ready for cranes to roost in. In this regard, the selected study site may offer a useful window to examine the effect of crane activities to the soil. After rice was harvested in September, 2020, a 3 m x 3 m of levee was established at the corner of the field (45 m x 10 m) and the area was covered with an anti-bird net to block the access of cranes (control). The rest of the field served as treatments, where cranes can come for food and shelter. Soil samples were collected at 0–15 cm from the control and the treatment  with a hand auger , before  and after  the migration of wintering cranes. Soils were homogenized with hand, and roots and rocks were picked manually. Before the migration, soil pH, gravimetric water content, and WEOC was 6.14 ± 0.08 (mean ± standard error), 33.21 ± 1.29 (%), and 51.47 ± 3.86  and two for the treatment plots , to monitor the presence of cranes between October, 2020 and March, 2021  over carboxylate  residues to estimate the quality of organic C. The lower, the more processed and more likely resistant to further decomposition [Total C and N content of soil was measured on 105°C dried, ground soil samples with an Elemental Analyzer . We quantified WEOC concentration by mixing 7 g of fresh soil with 35 mL of deionized water, shaking the mixture on an orbital shaker at 200 rpm for 4 h, filtering the mixture through a 0.45 μm filter  . CO2 concentration in the jar was recorded every 10 s for 10 min to generate a positive slope between CO2 concentration and time. The slope was used to calculate respiration rate at a given time (μg C-CO2 h-1 g-1 dry soil). We repeated measuring respiration rate every 30 min for 2 h . We refer this as basal respiration rate before adding substrates or adjusting water content and presented mean basal respiration [For basal respiration and substrate-induced respiration, we used methods described in Min et al. . Brieflypiration  (n = 4).piration , 44, 45.We extracted soil microbial DNA following the manufacturer’s instructions from the DNeasy Power Soil Kit . DNA extracts were stored at -80°C and sent to Chunlab Inc.  was checked for normal distribution (Shapiro-Wilk test) and has met the requirement for parametric analysis. We used a one-way ANOVA to test the effects of crane activities on soil chemical and biological properties. Independent variable was the existence of cranes and response variables were total C, total N, WEOC, the ratio of CH over COO (FTIR), DNA content, substrate-induced respiration, and basal respiration. We used R 4.0.2  and a stG.japonensis and 875 G.vipio in December 2020; 103 G.japonensis and 824 G.vipio in February 2021) were counted during the day in the broader rice fields including the study site. The number of the cranes roosting in the study site appears bigger: the census team observed at least 2,154 G.vipio on 15 Nov 2020, while local farmers claim 3,000–4,500 cranes staying overnight.Local farmers’ practices to flood the rice fields after harvest and to keep the rice straws within the rice fields have successfully attracted migrating cranes in the study site. Monthly crane surveys during 2020–2021 revealed that about 1,000 cranes  and dynamic (substrate-induced respiration) approaches  after thCrane activities not only increased soil microbial biomass, but also enhanced basal respiration rate . On averSoils under crane activities harbored more diverse microbial communities. We tested two Alpha diversity indices, Shannon index and the number of OTUs . ShannonBoth bacterial and fungal community composition was sensitive to crane activities, and microbial community composition significantly differed between the control and treatment soils Figs  and 7. FHere we provide evidence that the migration of cranes creates a system that differs in biogeochemical processes and biodiversity from surrounding environments. Below we discuss how migratory crane activities can drive distinct ecosystem processes via recharging, redistributing, and refreshing resources during winter.G.japonensis and G.vipio, respectively), we estimated how much of C and N are deposited into the soil system while cranes are present in the study site. Because the dropping weight is often 3% of the body weight [G.japonensis (c.9-10 kg) and 5,330 of G.vipio (c. 5-6kg) [In support of our first hypothesis, crane activities created distinct resource landscape in soils during winter compared to those without crane activities. Increases in total C and N in soils  could bey weight , it is e. 5-6kg) . Based o. 5-6kg) . Dead boIn addition to the changes in the resource quantity, we demonstrate that resource quality was also influenced by crane activities. DRIFT has been increasingly used in soil science to characterize soil C functional groups , 51–54. Alternatively, crane activities may have enhanced microbial decomposition of C in soil, decreasing the CH/COO. We found not only greater total  and metaTaken together, our results indicate that the crane migration elevated otherwise low resource availability and microbial activity during winter. We do not know if seasonal pulses of resources by crane activities would last until the following plant growing season, and if enhanced microbial biomass and activity would help plants grow better. Even though it is beyond the scope of the study, some studies imply that migratory birds can help achieve sustainable agriculture. For instance, Firth et al.  estimateOur second hypothesis that crane activities will enhance microbial diversity is supported in this study. There are at least three scenarios whereby the migration of cranes could increase microbial diversity. First, crane activities may have provided resources that were limiting to microbial growth. Soil microbial communities are often shaped by resource landscape and exhibit higher diversity at greater resource availability –57. As sConsistent with Fierer et al. , where NFor the past decades, rice fields have gained increased attention among South Korean environmental policy makers and practitioners, as viewed as a winter habitat for migratory birds, including endangered species such as cranes , 66. TheFor policy makers, the agricultural potential of crane activities can serve as an additional rationale to convince more farmers to join the biodiversity schemes in Cheorwon, and other rice fields used by migratory birds for wintering habitats. Farmers of Cheorwon have long been reluctant to join crane conservation. In addition to the perceived loss, they are concerned with the possibility that the presence of endangered species in the rice fields may lead to additional farming restrictions, and even the loss of their land by being designated as protected areas. The results of this study, however, suggest that inviting cranes to their rice fields can benefit not just the birds but farmers as well because the increased C and N in the soils could enhance the productivity of their rice fields. Specifically, if the reduction in the fertilizer use serves as incentive for farmers and if the incentive weighs over the costs to flood the fields and to keep rice straws, biodiversity conservation efforts could be more welcomed among local farmers. Furthermore, the reciprocity between the cranes and farmers disrupts the prevalent thinking that puts conservation and agriculture at the oppositional ends. Instead, it suggests the possibility of farmers’ living with the cranes through carefully designed conservation practices. In this regard, this study supports recent discussions of land sharing/sparing agriculture that try to find ways to reconcile agricultural land use with biodiversity conservation –70.The plight of the cranes caused by a series of anthropogenic intervention perhaps echoes the widespread biodiversity loss at the global scale, confirming the recent diagnosis of the Anthropocene. While scientific studies of the Anthropocene tend to focus on the effects of anthropogenic activities on the Earth, this study pays attention to the role played by nonhuman animals to intervene with the Earth’s surface. Our results demonstrate that animal migration is not a mere movement of biomass, but an ‘ecological force’ that shapes a distinct ecosystem with altered biogeochemical processes. We find that three months of crane migration was long enough to impact microbial biodiversity and soil C dynamics in agroecosystems. Crane conservation efforts in Korea have focused on the migration itself, but taking into account the ecosystem services that cranes can provide after their migration can help appeal to a broader people. The ecological benefits gained from crane activities could offer an additional rationale for expanding biodiversity protection schemes targeted at rice fields. Further studies are required to assess if changes in resource landscape and microbial activity/composition will promote plant growth, how resource landscapes will vary along with the annual cycle of crane migration, if the beneficial effects of crane activities will be proportional to the number of cranes or depend on the types of ecosystems/management history.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.


Citrullus lanatus) productivity in gravel-mulched fields were analysed for the following six fertilization modes: no N fertilizer input as a control (CK), N fertilizer without organic fertilizer (CF), and organic fertilizer replacing 25%, 50%, 75%, and 100% of mineral nitrogen . The results showed that, higher manure to nitrogen fertilizer ratios were positively correlated with the percentage of soil macroaggregates (> 0.25 mm), mean weight diameter (MWD), TOC and POC concentrations, and their ratios in different particle sizes. Compared with CF, the treatments with 50% to 100% organic fertilizer significantly increased TOC storage (5.91–7.84%) in the soil profile (0–20 cm). Moreover, the CF treatment did not increase SOC concentrations or TOC storage, compared with CK. The fruit yield (2014–2018) of watermelon significantly increased by an average of 31.38% to 45.70% in the treatments with 50% to 100% organic fertilizer, respectively, compared with CF. Our results suggest that the partial replacement of chemical fertilizer with organic manure  could increase the proportion of macroaggregates, POC and TOC concentrations, and TOC stock in aggregates with different particle size and improve the yield of watermelon in the gravel fields of arid northwestern China mulched with gravel and sand.Gravel and sand mulching is an indigenous technology that has been used for increasing soil temperature and improving crop yield and water use efficiency for at least 300 years in northwestern China. However, long-term application of inorganic fertilizer with gravel and sand mulch could decrease the soil organic carbon content, and how to improve soil fertility under gravel and sand mulching remains largely unknown. Thus, we evaluated the effects of the application of inorganic  and organic (manure) fertilizers on the distribution of soil aggregates and their associated organic carbon in a field mulched with gravel and sand. A 5-year (2014–2018) field experiment was conducted in the arid region of northwestern China. Total organic carbon (TOC), permanganate oxidizable carbon (POC), TOC reserves in soil aggregates with different particle sizes, and watermelon ( A layer of gravel and sand approximately 10 cm thick lies on the soil surface. Numerous studies have shown that gravel and sand mulching can reduce erosion from water and wind3, limit the accumulation of salt near the soil surface4, increase soil temperature, extend the whole growing season5, and retain moisture by increasing water infiltration and reducing evaporative water loss compared to bare soil surfaces6. Overall, the gravel and sand mulching facilitates microhydrological management and promotes increased crop biomass and crop yield 7. Given these beneficial aspects of gravel and sand mulching, it is unsurprising that its application is widely used in agriculture in arid and semiarid environments, such as in the United Arab Emirates8, New Mexico9, and Lanzorate of the Canary Islands10. At present, there are approximately 100,000 ha of gravel and sand-mulched fields in China, of which about 75% are used for growing watermelon (Citrullus lanatus) 1, which has become a highly economically lucrative crop in the arid northwest.Gravel and sand mulching has been used for at least 300 years in the arid area of northwestern China1. Unfortunately, the long-term application of inorganic fertilizer with gravel sand mulch could reduce the macroaggregate content as well as lower the mean weight diameter (MWD)11. Thus, gravel-mulched fields often show decreased Soil organic carbon (SOC) with continuous cropping11 and, consequently, degradation of other physical, chemical, and microbial properties14. In China, soil degradation in gravel-mulched fields in the arid northwestern region has significantly negative impacts on the watermelon crop.Despite the many benefits of utilizing gravel and sand mulching, it may also lead to soil deterioration. The addition of organic fertilizer would be a good method to improve soil fertilizer. However, the application of organic fertilizer, especially farmyard manure, involves many complex procedures due to the lack of special fertilization equipment, including removing the existing gravel mulch, spreading organic fertilizer, plowing, restoring soil surface uniformity, and re-mulching with gravel Fig. . In Chin15. It is an important index reflecting soil quality and soil health, which directly affects soil fertility and crop yield17. Lal17 found that the soil organic carbon pool increased by 1 mg ha−1 y−1 increasing food grain yield by 32 million mg y−1 in developing countries. Studies have found that SOC is mainly distributed in macroaggregates (> 1 mm), and its content increases with aggregate diameter19. The microaggregates (< 0.25 mm) can effectively reduce the loss of SOC, and accumulation of greater SOC in microaggregates effectively promotes macroaggregates21. Meanwhile, SOC accumulation improves soil aggregation, yielding an even greater capacity for SOC storage22. Thus, this positive interaction between SOC and aggregate formation achieved a physical protection of SOC. In recent years, numerous studies have found nutrient management is an important factor governing SOC and aggregates in soils31. Moreover, the application of organic materials or combining organic fertilizer and inorganic fertilizer have significantly positive effect in increasing the SOC content, improving macroaggregates formation and enhancing the stability of soil aggregates31.Soil organic carbon (SOC) serves as an important sink for many nutrients, drives the soil nutrient cycle, helps to maintain structural stability, aids in air and water infiltration, promotes water retention, and reduces erosionHowever, few prior studies have been conducted to investigate the potential of replacing chemical fertilizer with manure in soil restoration and maintenance in gravel-mulched watermelon fields within the sandy, arid region of northwestern China. Therefore, in this 5-year study, we partially replaced chemical fertilizer with manure in a 20-year-old gravel-mulched watermelon field in northwestern China with the objectives of evaluating the effects on fruit yield of watermelon, sizes and abundance of soil aggregates, and relative distribution of SOC and SOC stocks retained by the different aggregate sizes. We expect that our results have certain guiding significance for soil quality improvement and the optimization of watermelon nutrient management strategies in gravel-mulched fields in northwestern China.This 5-year study 2014–2018) was conducted on at experimental site established by the Gansu Academy of Agricultural Sciences (GAAS) Gaolan Research Station of Ecology and Agriculture , Gaolan County, Lanzhou city, Gansu Province, China. The site has an elevation of 1830 m above sea level  cultivar, 'Xi Sha Rui Bao', was used as the experimental material. The inorganic fertilizer used in this experiment included urea , calcium superphosphate  and potassium sulfate . The organic fertilizer used in this experiment was cow manure, and its nutrient content was determined by conventional chemical analysis methods 32.The watermelon .No nitrogen fertilizer  applied in accordance with the optimized amount of fertilizer selected from the preliminary work of the project.Inorganic fertilizer : 75% of the total N-fertilizer was supplied by inorganic fertilizer, and 25% was supplied by cow manure.Organic fertilizer replacing 25% of mineral nitrogen : 50% of the total N-fertilizer was supplied by inorganic fertilizer, and 50% was supplied by cow manure.Organic fertilizer replacing 50% of mineral nitrogen : 25% of the total N-fertilizer was supplied by inorganic fertilizer, and 75% was supplied by cow manure.Organic fertilizer replacing 75% of mineral nitrogen : all of the total N-fertilizer was supplied by cow manure.Organic fertilizer replacing 100% of mineral nitrogen , watermelon seeds of ‘Xi Sha Rui Bao’ were planted in the middle of April, and harvested in the middle of July. The row spacing was 1.0 m, the distance between holes was 0.6 m, and one seed was planted in each hole, representing a total of 11,335 plants per hectare. Before sowing, all of the organic fertilizer (cow manure), 30% of the total inorganic N-fertilizer, all of the inorganic P-fertilizer, and 40% of the total inorganic K-fertilizer were applied as basal fertilizer at a depth of 20 cm to the planting row of watermelon according to the steps shown in Fig. 4–2018, w33. Then, these air-dried samples were divided into two subsamples: one subsample was used to analyse soil aggregates and their associated organic carbon, and one subsample was used as a backup.In 2018, after the final watermelon harvest, we collected 3 soil samples under the gravel mulching layer in each planting row. The soil samples consisting of 10 cm × 10 cm in sections at a depth of 20 cm were randomly collected using a spade. Then we transported the samples in hard plastic boxes from the field to the laboratory to preserve the original physical structure of the soil. In the lab, the collected fresh soil samples were air-dried at 4 °C according to the method of Dorodnikov et al. (2009) so that the soil moisture of all samples reached 10–12%34. The air-dried soil sample was broken into soil particles < 5 mm along the natural textures. These 100 g soil particles were placed on the uppermost layer of a series of sieves  in an automatic sieve shaker . After 2 min, aggregates of > 2 mm, 1–2 mm, 0.25–1 mm and < 0.25 mm were achieved under the condition of 1.5 cm amplitude and 30 times/min frequency. Then, the mass of the agglomerates with the corresponding size class was obtained by weighing. We used the resulting dry mass from the sieves to compute the mean weight diameter (MWD) asi is the mean diameter of aggregates in a sieve fraction (i) in mm, and Wi is the proportion of the total sample mass represented by the sieve fraction.The stability of the air-dried soil aggregates was determined according to the method of Kemper and Rosenau (1986)35. Moreover, to measure permanganate oxidizable carbon (POC) concentrations in the aggregate fractions, we used the permanganate oxidation method with oxidation by 333 mmol L−1 KMnO4 measured with a spectrophotometer at 565 nm36.For the aggregate fractions from each sieve, we also analysed total organic carbon (TOC) concentrations using the dichromate oxidation method with external heating37.i is the percentage represented by a sieved fraction (i) and Ci is the percentage organic carbon content represented by the fraction.The contribution rate of aggregates to organic carbon (CAOC) was calculated according to the formula of Li et al. (2014)−1) in different aggregate fractions using the following equation38:−3).We determined the TOC storage , we calculated yield results as kilograms of fruit biomass per plot and extrapolated them to ha, expressed as kg per ha.We harvested watermelon from each 64 mData were statistically analysed using analysis of variance (ANOVA). The significance of the treatment effects was analysed using SPSS 17.0. Variance analysis and mean values (n = 3) were compared with the least significant difference (LSD) at the 5% level.Guideline statement: The watermelon cultivar ‘Xi Sha Rui Bao’ is not endangered species, and the cultivation and collection of plant material, field studies and laboratory experiments are all in compliance with relevant national regulations and legal regulations.0.25) and MWD increased by 8.26–18.20% and 14.67–33.33% in the OF-50%, OF-75%, and OF-100% treatments, respectively, relative to CF. Thus, increased application rates of organic manure were correlated with an increase in macroaggregates and MWD.The proportion of large macroaggregates (≥ 1 mm) significantly decreased with decreasing particle size, but small aggregates (< 1 mm) increased with particle size Table . The difTOC concentrations increased with decreasing particle size, with the greatest TOC levels in the < 0.25 mm fraction Table . The TOCThe POC concentration followed a similar trend to that of TOC; namely, it increased in different-sized aggregate fractions with the increasing application rate of organic manure. We observed the greatest level of POC in the macroaggregate fraction of 0.25–1 mm  than microaggregates (< 0.25 mm) with the highest values in the 0.25–1 mm aggregate fraction Table . The perThe contribution to TOC of the > 1 mm aggregate fraction (CAOC) decreased with decreasing particle size and increased with increasing application rates of organic manure Table . In the TOC storage increased with decreasing particle size in the ≥ 1 mm or < 1 mm aggregate fractions and increased with increasing application rates of organic manure, with the maximum value in the < 0.25 mm fraction Table . HoweverThe fruit yield of watermelon increased with increasing application rates of organic manure and was significantly higher in the OF-75% and OF-100% treatments Fig. , which i40. Obviously, both water infiltration and retention are critical within gravel-mulched fields in the arid loess region of northwestern China. Therefore, the stability of soil aggregates under water influx may be a more sensitive indicator than the properties of bulk soil strength of changes to soil quality and function induced by fertilizer management in the gravel-mulched fields of the region41.When macroaggregates of soil are stable in water, they reduce surface crusting and help to control soil erosion by water and wind, thus improving infiltration of water and water retention31, who showed that long-term application of organic fertilizer and the combined application of organic and inorganic fertilizers significantly promote the aggregation of macroscopic aggregates (> 0.25 mm) compared with inorganic fertilizer.Over the 5-year duration of this study, we found that the application of inorganic N fertilizers had no significant effect on the formation and stability of soil aggregates, relative to no N fertilizer. In contrast, the application of organic manure substantially increased the proportion of the macroaggregate fractions (> 1 mm) when compared with soils that received no organic manure, and this is consistent with the results from Ma et al. (2022)28. The promotion of macroaggregates under an organic fertilizer regime may be related to the accumulation of humic substances, which result from the breakdown of organic matter and increase following the application of organic fertilizer. Tang et al. (2018) reported that soil humic acid (C-HAF), fulvic acid (C-FAF), and humin (C-HUM) increased under the long-term application of a combination of manure and mineral fertilizer26. We found that the macroaggregate fractions (R0.25) and MWD increased with increasing application rates of organic manure and that the microaggregate fractions decreased. Thus, our results support prior studies indicating the advantage of organic manure fertilizer in macroaggregate formation and, therefore, its capacity to improve soil structure and, consequently, soil function.In addition, macroaggregate stability in soil is promoted by the application of organic manure fertilizer. Chen et al. (2020) reported that long-term, excessive application of chemical fertilizer combined with sustained mono-cropping of tobacco reduced water-stable soil aggregate stability, while the use of organic fertilizer increased the proportion of large macroaggregates and MWD and significantly reduced the proportion of microaggregates42. However, the correlation of N to SOC remains incompletely understood. Some studies indicate that the application of nitrogen fertilizer can significantly increase the SOC content in soil, and its application rate had a positive correlation with SOC concentration 43. Moreover, in China, Yang et al. (2003) reported that inorganic-N and NPK fertilization were inadequate for maintaining SOC levels under conventional management of corn with no aboveground crop residues returned to the soil44. In contrast, Russell et al. (2005) reported that N fertilization negatively impacted SOC by the density of edaphic bacterial communities45. Our study showed that inorganic N applied at rates of 200 kg N per ha (CF treatment) yielded higher SOC contents than the control , but the difference was not significant. However, we found that SOC had significant improvement in different-sized aggregate fractions when 50% and more N was replaced with manure than in the CK and CF treatments  and summer maize (Zea mays L.), SOC significantly increased by 70.7–121.7% over the initial, pretreatment measurement, while inorganic fertilizer produced an increase in SOC of only 5.4–25.5%, which was not significant over the same timeframe47. Similarly, in the rice barley cropping system of eastern China, a 23-year manure application increased the soil SOC content by 25%, while inorganic fertilizer application only increased by 13%23. These prior results are highly comparable to ours, in which we found that the application of manure fertilizer yielded overall higher SOC concentrations in differently sized soil aggregates compared to inorganic fertilizer alone, and that the rates of SOC increase were positively correlated with the proportion of organic to inorganic fertilizer applied.Many long-term field experiments have shown that a hybrid fertilization strategy comprising the application of both inorganic and organic fertilizers can increase soil nutrient and SOC concentrations48. However, other studies suggest that the stability of SOC is greater in smaller aggregates 50 because the smaller aggregates represent more of a closed system affording greater physical protection 52. We propose that the reasons for discrepancies among prior studies may be because that the decomposition of abundant cellulose and sucrose in macroaggregates temporarily leads to an increase in SOC 53. Decomposition of cellulose and sucrose in macroaggregates may also occur more quickly due to the favourable biotic (and enzymatic) environment of macroaggregates47. Our data are congruent with those of Puget, et al. 49 and Ashman, et al. 50 in indicating that microaggregates (< 0.25 mm) had the highest TOC content, possibly because newly added C from manure becomes robustly sequestered within the microaggregates.The relationship between soil aggregate sizes and SOC concentrations remains poorly understood. Some previous studies have found that macroaggregates have a higher concentration of SOC than microaggregates possibly because macroaggregates are less likely to fully break down and release C under stress2. We found that POC and the ratio of POC to TOC were improved under increasing application rates of organic manure. Among differently-sized soil particles, we found that macroaggregates (> 0.25 mm) had higher POC:TOC ratios than microaggregates (< 0.25 mm), which might be attributable to the unfavourable environment for microbially facilitated transitions from inactive to active carbon within the microenvironments of microaggregates47. Similar to our results, Tong et al. (2020) also found that the application of organic fertilizer significantly increased soil POC among different-sized soil aggregates54.Permanganate oxidizable carbon (POC) is the active part of SOC that, following processing by microorganisms, can be readily used by plants. Qiu et al. (2015) reported that active carbon (permanganate oxidizable carbon) was the best overall indicator of the soil C status and decreased with increasing cultivation time in gravel-mulched fields55. Taken together with our results, this finding supports that a greater abundance of macroaggregates facilitates a higher concentration of SOC. Moreover, we found that TOC storage and the CAOC of macroaggregates increased with increasing application rates of organic manure. Similarly, Su, et al. 56 determined that approximately 57–64% of the total SOC was stored in macroaggregates (> 0.25 mm) after treatment with organic fertilizer, compared to 54–60% prior to treatment.The distribution of soil aggregate sizes and their differing organic carbon content (CAOC) objectively reflect the effect of fertilizer management practices on the organic carbon pool of soil. Our results showed that macroaggregates (> 0.25 mm) had higher CAOC than microaggregates (< 0.25 mm), despite microaggregates having higher TOC. As a result, macroaggregates (> 0.25 mm) had greater TOC storage than microaggregates (< 0.25 mm), and approximately 78% of the total SOC was stored in macroaggregates (> 0.25 mm). This finding is consistent with the results of Li et al. (2013), who reported that macroaggregates contained more SOC than microaggregates and that there was a positive relationship between the abundance of macroaggregates and total SOC57. Zhou et al. (2013) showed that wheat biomass had a significant correlation with SOC and soil total N in the 0–20 cm soil layer, and mineral fertilizers applied together with cattle manure significantly increased the SOC and total N content in the topsoil layers and dramatically improved winter wheat grain and aboveground biomass yields58.The application of organic fertilizer improved the soil physical structure and chemical properties by increasing soil aggregation and promoting soil stability. The combined application of inorganic and organic fertilizers likely reduced water loss and improved the efficiency of soil hydrological cycling. Improvements in soil hydrology and watermelon yield under organic fertilization regimes were also observed by Du et al. (2020), who reported that organic fertilizer application increased soil water use efficiency by 54.9–176.3%, N-use efficiency in watermelon by 6.9–18.5%, and the watermelon yield by 63.2–156.6% 59. A balance of fertilization with both organic manure and inorganic NPK may yield changes in the soil microbial composition, especially reducing fungal abundance while increasing bacterial abundance, which promotes the soil cycle and healthy soil structure 60.The application of organic manure over a 5-year period reduced the typical soil-depletion effects of continuous cropping in a gravel-mulched field. Overall, improvements to the soil quality are likely related to the effects of manure application on the structure and function of the edaphic microbial community This study showed that the fruit yield of watermelon increased with increasing application rates of organic manure over 5 years in a gravel-mulched field. We suspect that there are two main reasons for this result:In general, our results support the incorporation of both inorganic and organic fertilizers as part of an integrative soil fertility management strategy for improving the watermelon yield.The partial replacement of chemical fertilizer with organic manure significantly improved the proportion of macroaggregates as well as the SOC content of different -sized aggregates in the topsoil and, consequently, increased watermelon yields relative to chemical fertilizer alone. Long-term application of inorganic fertilizers is inadequate for maintaining SOC and soil nutrient levels, especially under conventional management strategies of gravel-mulched fields. In contrast, the partial or complete replacement of chemical fertilizer with organic manure may recover and maintain soil quality, thus overcoming this constraint on the utilization of gravel-mulched fields. Our work has implications for the sustainable development of the watermelon industry and protection of the natural and agroecological environment in the Loess Plateau of arid northwestern China.Supplementary Information.


Drought is one of the main concerns worldwide and restricts the development of agriculture. Silicon improves the drought resistance of plants, but the underlying mechanism remains unclear.Prunus persica) seedlings were used to analyse the effects of silicon on plant growth and physiological indexes related to drought resistance under drought stress. The results showed that silicon addition improved the water use efficiency, antioxidant capacity, and net photosynthetic rate, inhibition of stomatal closure, promoted the development of roots, and further regulated the synthesis of hormones, amino acids and sugars in peach seedlings. A comparative transcriptome analysis identified a total of 2275 genes that respond to silicon under drought stress. These genes were mainly involved in ion transport, hormone and signal transduction, biosynthetic and metabolic processes, stress and defence responses and other processes. We analysed the effects of silicon on the modulation of stress-related hormonal crosstalk and amino acid and sugar metabolism. The results showed that silicon promotes zeatin, gibberellin, and auxin biosynthesis, inhibits the synthesis of abscisic acid, then promote lateral root development and inhibit stomatal closure, and regulates the signal transduction of auxin, cytokinin, gibberellin and salicylic acid. Silicon also regulates the metabolism of various amino acids and promotes the accumulation of sucrose and glucose to improve drought resistance of peach seedlings.We sequenced the transcriptomes of both control and silicon-treated peach seedlings under drought stress to identify genes or gene networks that could be managed to increase the drought tolerance of peach seedlings. Peach (Silicon enhanced the drought resistance of peach seedlings by regulating stress-related hormone synthesis and signal transduction, and regulating amino acid and sugar metabolism.The online version contains supplementary material available at 10.1186/s12870-022-03785-5. With global climate change, the growth and development of plants are often subject to different environmental pressures, such as drought, heat, cold, and high salinity . In partThe root system of peach trees is shallow, and drought stress will adversely affect peach production. Drought stress is one of the major abiotic stresses limiting fruit production and quality during the 4–6 week period before harvesting . And droDrought stress can cause a series of physiological and biochemical reactions in plants, such as the limitation of photosynthesis, enhanced stomatal closure, changes in the cell composition, stimulation of osmotic fluid, and accumulation of reactive oxygen species . Under dThe content of silicon in the Earth’s crust is close to 28%, which is the second most abundant element in the Earth’s crust . The con2− and H2O2 could be due to the Si triggered decline in oxidative burst by lessening ion toxicity and accretion of nucleoproteins that provide protection to plants against stress conditions [Silicon alleviates various types of abiotic and biotic stresses, such as drought, cold, salt, heavy metals, plant diseases and insect pests , 25. Undnditions . The resnditions , 35. Silnditions . Siliconnditions .Prunus persica (L.) Batsch) is a significant fruit crop worldwide and the fourth most extensively produced fruit crop in China. In this study, we found that exogenous silicon can enhance the drought resistance of peach seedlings and thus performed RNA-Seq to analyse the transcription changes of peach seedlings after exogenous silicon treatment under drought stress. Our analysis showed that silicon changed the transcriptome of peach seedlings under drought stress and enhanced the drought resistance of peach seedlings by regulating hormone, amino acid, and sugar metabolism. Silicon may be an elicitor of drought tolerance in peach seedlings. This research might provide a theoretical foundation for the use of silicon in crop production under drought conditions.Peach  Batsch) seedlings were used as the test materials, which has high genetic stability, and the tests were performed at the experimental station of Shandong Agricultural University in 2021. Peach seedlings (15 leaves) with consistent growth and that were free of disease were selected for short-term drought stress experiments. Three treatments were set up in the experiment: control , drought treatment , and drought treatment plus the application of silicon . A PEG6000 solution was used to simulate drought, sodium molybdate was added to the solution, and the molybdenum concentration was 0.6 mmol/L. For each treatment, 18 seedlings were selected and cultivated in a hydroponic tank equipped with different treatment solutions for 6 h. After the treatment, the morphology of the seedlings was observed, the water use efficiency was determined, the roots and leaves were stained with Evans blue, and samples were collected to determine the antioxidant enzyme activity.Peach  with consistent growth and that were free of disease were selected for the continuous drought stress test. Three treatments were set up in the experiment: control , drought treatment (10% PEG), and drought treatment plus the application of silicon (10% PEG + Si). For each treatment, 30 seedlings were selected and planted in pots with quartz sand in a greenhouse with light  and day/night temperatures of 28 °C/18 °C, and the air humidity is 50%. Drought stress was carried out by controlling the water content of quartz sand in the pots, water content adjustment with 10% PEG solution. The water content of the CK treatment was 60%, and the water content of the PEG% and PEG% + Si treatment groups was 40%. The application of silicon was 0.6 mmol/L NaAfter 10 days of continuous drought stress treatment, samples were collected from the drought-treated and drought + silicon-treated groups for RNA-Seq. Peach seedlings that randomly selected were rinsed with sterile water, and leaves and roots were clipped for analysis. Three peach seedlings from each treatment were blended as a biological replicate, and 3 replicates of each treatment were included in the study. The samples were promptly frozen in liquid nitrogen and maintained at a temperature of -80 °C until analysis.Evans blue staining examination has been confirmed as a reliable method for microscopic cell death determination. We used that method to determine cell death, as previously described by Baker and Mock, with slight changes. Nine root tips and leaves from each treatment were selected for staining. samples were vacuum infiltrated in 0.1% Evans blue (w/v) solution for 5 min before staining at room-temperature for 3 to 5 h. The samples were then rinsed in phosphate-buffered saline (PBS) containing 0.05% (v/v) Tween-20. The image was taken using an electron microscope. A darker root and leaf image indicated a lower cell activity.On a sunny day, the water use efficiency and net photosynthesis of fully developed functional leaves were measured using the CIRAS-3 portable photosynthetic measurement system . These measurements were repeated 6 times, and the results were averaged.The functional leaves with the same leaf position were cut, weighed and placed in the room, and weighed once at a fixed time to calculate the percentage change rate of leaf weight. Three replicates were performed for each treatment, and the results were averaged.For analysis of the root architecture, the peach seedlings were completely removed, and the roots were thoroughly rinsed. Root architecture parameters  were measured using the Professional WinRHIZO root analysis system (Epson V 700).The content of superoxide anion was determined by hydroxylamine oxidation method; the enzyme activity of superoxide dismutase was measured by nitroblue tetrazolium photoreduction method; the activity of peroxidase was measured by guaiacol method; the activity of phenylalanine ammonia lyase was measured by L-phenylalanine method ; silicon10 days after processing, three leaves of the same developmental stage were selected to determine the size of the stomata. Take the lower epidermis of leaves to make specimens, then observed under 400 × magnification using a Fluorescence Microscope .Abscisic acid, zeatin, auxin, and gibberellin were extracted from the freeze-dried samples and measured . The ext−1 for 2 min, and the supernatant was collected. (3) The sample was passed through a 0.22-μm water filter membrane, and 1 mL of the sample was collected.For analysis of the amino acid composition, the contents of amino acids were determined using a fully automatic amino acid analyser . According to the literature , the samIn the analysis of the sugars and acids contents, the levels of glucose, fructose, sucrose, citric acid and malic acid were determined by capillary zone electrophoresis (CZE), as previously described . The ext−ΔΔCT approach [According to the manufacturer’s instructions, an RNAprep Pure Plant Kit  was used for the isolation of total RNA from 0.5 g of samples. The PrimeScript™ RT Kit  was used to generate first-strand cDNA according to the manufacturer’s instructions. With a QuantStudio® 3 real-time PCR instrument  and three biological and three technical duplicates, we performed a RT-qPCR analysis using SYBR® Premix Ex Taq™  and PpActin as the reference gene in accordance with the manufacturer’s recommendations for all reagents. The relative expression levels were calculated using the 2approach .TRIzol reagent was used for the extraction of total RNA according to the manufacturer’s instructions. A NanoDrop 2000 spectrophotometer was used to assess the RNA purity and quantity . An Agilent 2100 Bioanalyzer was used to check the RNA integrity . Libraries were then constructed using the TruSeq Stranded mRNA LT Sample Prep Kit  according to the manufacturer’s instructions. OE Biotech Co., Ltd. Performed the transcriptome sequencing and analysis .P value < 0.05 and fold change > 2 or < 0.5. To show the expression pattern of genes in the various groups and samples, a hierarchical cluster analysis of differentially expressed genes (DEGs) was performed. Based on the hypergeometric distribution, GO enrichment and KEGG pathway enrichment analyses of the DEGs were performed using R.The libraries were sequenced using an Illumina HiSeq X Ten platform, and 150-bp paired-end reads were produced. The clean reads were mapped to the peach genome  using HISAT2 . The FPKThe biological and biochemical data are presented as the means ± standard errors (SEs). The significance of the differences between samples was assessed by Duncan’s multiple range tests using SPSS version 20.0. Separation of signifcant means was accomplished using Duncan Multiple Range Test at 0.05 signifcant levels. One-way analysis of variance (ANOVA) was carried out for statistical analyses of multiple experimental groups.Under drought stress, the leaf water content of plants decreases with increases in the stress intensity, and the water content can reflect the drought resistance ability of the plants to a certain extent. As shown in Fig. The leaves and roots of the seedlings were further analysed and stained with Evans Blue. The staining degree of the leaves and roots of the seedlings treated with silicon under drought stress was significantly lighter than that of the seedlings subjected to the drought treatment Fig. b and c, To further study the role of silicon in drought, we subjected peach seedlings to continuous drought treatments to observe the effects of exogenous silicon treatment on their growth. After 10 days of treatment, we found that exogenous silicon treatment increased the drought resistance of peach seedlings, and further observed the stomata of leaves. Microscopic observations revealed that drought stress caused the leaf stomata to close. Based on the photographs, drought treatment closed the peach leaf stomata Fig. a, decreaAn analysis of the underground parts of peach seedlings revealed thatdrought inhibited the growth of plant roots and reduced root activity Fig. , and exo2SiO3 to study the genetic basis for the drought tolerance of peach seedlings exposed to exogenous silicon through RNA-Seq analysis. The RNA-Seq data from three biological duplicates of each sample yielded an average of 44–50 million rawreads. We acquired an average of 43–49 million clean readsafter filtering out low-quality reads; more than 92% of the sequencing quality was Q30, and the average GC content of each sample was approximately 46%. We were able to correctly map 92% of the genes to the peach reference genome, and these included more than 90% unique mapped reads and fewer than 2.7% duplicated mapped reads  of the transcriptome data, and based on the FPKM values, we calculated Pearson correlation coefficients (PCCs) among all expressed genes in all the samples Fig. a and b. To better understand the function of each DEG, a GO enrichment analysis of the DEGs was performed, and their functions were described. The results showed that a total of 2023 DEGs were annotated with 2023 GO terms, and we screened and classified these terms. According to the functional classification, we divided the GO into three levels: level 1, level 2 and level 3. Level 1 contains three items, namely, biological process, cellular component, and molecular function; level 2 contains 64 items, including biological adhesion, cell, and binding; and level 3 contains tens of thousands of items used for conventional enrichment. According to the top 30 GO enrichment terms , carotenoid biosynthesis (ko00906) and glutathione metabolism (ko00480) Table S. Thirty-The effect of exogenous silicon on the resistance of peach seedlings involves the hormone pathway. Thirty-nine DEGs implicated in hormone production and signal transduction were discovered and investigated. The 21 DEGs involved in hormone synthesis were found to be related to the synthesis and decomposition of zeatin, abscisic acid, gibberellin, auxin and cytokinin Table , which aThe KEGG enrichment analysis showed that the regulation of silicon on the drought resistance of peach seedlings involves amino acid metabolism, metabolism of other amino acids, glycan biosynthesis and metabolism and carbohydrate metabolism. Thus, we compared the contents of various amino acids and carbohydrates in peach seedlings under drought stress. The results showed that drought stress caused significant changes in the amino acid contents of peach seedlings, increased the total amino acid content, and upregulated the contents of aspartic acid, glutamic acid and proline in leaves and roots. Compared with the results obtained with the drought stress treatment, silicon increased the contents of aspartic acid, proline, glycine, phenylalanine, tyrosine and other amino acids in peach seedlings Fig. a and b. To test the reliability of the RNA-Seq data, 12 DEGs were selected for qPCR analysis using TMV resistance protein N (LOC18788576) as a reference gene. The representative genes selected for qPCR analysis were genes involved in hormone biosynthesis, hormone signal transduction, amino acids, starch and sucrose metabolism  and the biosynthesis of compatible solutes [Water deficits affects the growth of plants, and plants can maintain their water balance and improve their resistance by increasing the absorption of inorganic substances (usually K solutes . The exp solutes . The tol solutes . Under d solutes , 64. Exo solutes . In summAs an effectual quasi-essential element, silicon plays an important role in improving the drought resistance of peach seedlings. This study showed that exogenous silicon alleviates the damage to peach seedlings induced by drought stress as well as the adverse effects on growth and development, the increases in oxidative damage, the acceleration of water loss, and the decrease in photosynthesis caused by drought. Silicon reduces drought stress damage through various mechanisms, including increasing the accumulation of silicon in peach seedlings, improving antioxidant enzyme activity, improve plant water use efficiency, regulating the synthesis of compatible solutes, mediating hormone synthesis and signal transduction pathways, regulating the ratio and balance of hormones. Our results show that silicon increases the drought resistance of plants through a variety of physiological and biochemical reactions.Additional file 1: Table S1. Mapping statistics of the RNA-Seq data for three replicates in each treatment. Table S2. DEGs involved in ROS scavenging. Table S3. DEGs involved in hormone signal transduction. Table S4. DEGs involved in amino acids metabolism. Table S5. DEGs involved in starch and sucrose metabolism.


African men are less likely to participate in prostate cancer (PCa) screening, which may be beneficial to some of them. Gaps in knowledge, cultural factors and beliefs are associated with their screening intentions.To determine the knowledge, cultural factors and screening intentions of African males regarding PCa screening.The study was conducted among African men attending randomly selected primary healthcare clinics in the Free State province.An analytical, cross-sectional survey using self-administered questionnaires developed in line with the Theory of Planned Behaviour constructs.p < 0.001), higher perceived benefits of PCa screening , lower perceived situational barriers to PCa screening  and higher perceived risk of getting PCa .Of the 389 respondents, 18.3% had ever been screened for PCa with prostate-specific antigen (PSA) testing and 6.2% by digital rectal examination (DRE). About a quarter (24.4%) of the respondents had knowledge scores ≥ 50%. Factors associated with greater intent to screen for PCa were lower degree of fear/apprehension of PCa screening (mean score = 2.03; The observed low level of knowledge and practice of PCa screening among the respondents could be enhanced through PCa awareness strategies targeted at these men or those that could influence their decision making, especially healthcare providers. Factors that enhance screening intentions should be promoted.This study improves on the scarce literature on factors associated with African men’s PCa screening intention. Prostate cancer (PCa) remains a substantial healthcare burden. The global incidence of PCa was the second highest after lung cancer in 2020  is controversial because of the associated overdiagnosis and overtreatment. However, more recent studies reveal that the net benefit of PSA screening may be beneficial to black men compared with the general population  with a life expectancy of more than 10 years if they have a family history of prostate or breast cancer in a first-degree relative, lower urinary tract symptoms and/or clinical suspicion of PCa, regardless of age group , theory of reasoned action (TRA), socio-ecological model (SEM) and health belief model (HBM) have engaged in understanding patients’ behaviours and decisions regarding health practices. The present study adopts the TPB to achieve the study objectives  the background characteristics of African men in the Free State, (2) their level of knowledge and the knowledge gaps regarding PCa and PCa screening, (3) their culture and beliefs regarding PCa screening and (4) their screening intentions. The secondary objective was to determine the factors that impact their screening intentions for PCa.This study was an analytical cross-sectional survey of African men attending selected primary healthcare (PHC) clinics.The target population of the study included African men aged 40–70 years in the Free State.Extrapolating from data available from StatsSA 2019 . It was impractical to visit all the clinics because of financial and time constraints. The list of these clinics was obtained from the respective district offices. Through stratified simple random sampling method, 115 of these 231 fixed PHC clinics were selected . Four respondents were targeted per clinic, giving a total of 460 respondents targeted.To avoid introduction of bias because of prior knowledge, those already diagnosed with PCa or benign prostatic hyperplasia and those who have had prostate investigations, such as biopsy or ultrasound, were excluded from the study.A self-administered survey questionnaire was used. With the assistance of language translators from the University of the Free State (UFS), the questionnaire was translated from English into Sesotho and Isizulu, which are the common local languages Wazimap  in the aThe questionnaire adopted for this study was a validated modified version of the Thomas Jefferson University Prostate Cancer Screening Survey Questionnaire, which was developed using the constructs of the TPB Kenerson .The questionnaire consisted of six sections: Sections A - F.Section A  containeTrue, False or I don’t know options.Section B  containeBloom’s cut-off points moderate knowledge (60% – 79% correct responses)poor knowledge (< 60% correct responses).Section C consisted of the eligibility criteria for participation in the PCa screening survey. To be eligible, the respondents must: (1) be 40–70 years of age, (2) be African by race, (3) have heard about PCa, (4) not have been previously or currently diagnosed with PCa and (5) not have had a prostate biopsy in the past.Section D was the PCa screening survey. This section explored the cultural factors  likely to be associated with respondents’ intent to screen for PCa. Total subscale scores were generated for fatalism, fear/apprehension, perceived benefits and subjective norms before statistical inferences were tested.Section E explored the respondents’ source(s) of knowledge about PCa and PCa screening, while Section F checked the respondents’ perceived need for more knowledge about PCa and PCa screening and their preferred method of receiving such knowledge.Constructs of the TPB were operationalised to examine the role of social and cultural determinants of PCa screening behaviours among African men.This is the degree to which PCa screening is positively or negatively valued. Attitude and beliefs were operationalised by measuring fatalistic views of PCa, PCa screening, fears/apprehension and the perceived benefits of PCa screening.This represents perceived social pressure to adhere to PCa screening. Normative beliefs were operationalised by measuring the social influence of relevant other(s)  on PCa screening behaviours.Situational barriers are factors that hinder the decision to participate in PCa screening. The situational barriers assessed in this study included concerns about cost, time commitment, embarrassment and pain associated with PCa screening.The intent was operationalised by measuring the intention to participate in PCa screening within a six-month period.These are factors that may or may not influence the intention to participate in PCa screening tests. Such factors include demographic characteristics, PCa screening history, family history (first-degree relatives) of PCa, perceived risk of PCa and socio-economic status of respondents.= strongly disagree, 2 = sort of disagree, 3 = sort of agree and 4 = strongly agree).Items relating to attitude, subjective norms, situational barriers, screening intent and contributory or demographic factors as listed above were measured on a four-point Likert scale . Descriptive statistics (e.g. median and standard deviation [SD]) were used for continuous variables, while frequencies and percentages were computed for categorical data. Association between variables was assessed using chi-squared or Fisher’s exact tests. A This study was approved by the Health Sciences Research Ethics Committee of the Faculty of Health Sciences, UFS, with ethical clearance number: UFS-HSD2020/1481/2411. Permission to conduct the study was obtained from the head of the Free State Department of Health.Following a detailed description of the study, signed informed consent was obtained from each respondent before they participated in the study. The self-administered questionnaire was anonymous as no names of respondents were recorded on any of the documents.A total of 389 men participated out of the 460 eligible respondents invited, giving a response rate of 84.6%. The median age of the respondents was 48 years (range 40–70 years). Only four of the respondents’ relatives had PCa (one father and three brothers).n = 188, 48.3%) of the respondents had never had a PSA test, 130 (33.4%) did not know about the test, 42 (10.8%) had the test over a year ago, while 29 (7.5%) had the test within the past one year. For the 71 respondents who had PSA testing in the past, their perception of the healthcare providers’ conduct of shared decision-making was assessed  of the respondents had never had a digital rectal examination (DRE), 158 (40.6%) did not know about the DRE, 16 (4.1%) had a DRE over a year ago, while eight (2.1%) had a DRE in the past one year.The majority (Only 53 (13.6%) respondents felt that they were at risk of getting PCa.n = 217, 55.8%), private general practitioners , spiritual healers  and traditional healers .The respondents’ preferred (first choice) healthcare providers were state PHC providers  had poor knowledge (i.e. scored < 60%). However, 95 (24.4%) scored ≥ 50%. The mean knowledge score was 5.67  out of 16 points. Based on the Bloom’s cut-off points, the majority of respondents , indicating that these respondents held little fatalistic beliefs related to PCa and PCa screening. Examples of fatalism items include ‘If I am meant to get prostate cancer, I will get it no matter what I do’ and ‘If I get prostate cancer, nothing can be done to cure me of the disease’. The ‘fear/apprehension’ mean score was 2.03 (± SD 0.69), indicating a low degree of fear/apprehension associated with PCa and PCa screening among these respondents. Examples of fear/apprehension items include ‘I am bothered by the possibility that prostate cancer screening might be physically uncomfortable’ and ‘Men who go through prostate cancer screening will have more problems than men who do not go through screening’. The ‘perceived benefits’ of screening’s mean score was 2.69 (± SD 0.60), indicating moderate beliefs in the benefits of screening among these respondents. Examples of perceived benefit items include ‘I believe that going through prostate cancer screening would help me to be healthy’ and ‘I believe that I can protect myself from prostate cancer by going through screening’.The ‘social influence’ mean score was 2.90 (± SD 0.72), showing a moderate perceived level of influence that family members and healthcare providers have on PCa screening among these respondents. Examples of subjective norm items include ‘I want to do what the doctor I see thinks I should do about prostate cancer screening’ and ‘Members of my immediate family are likely to think I should go through prostate cancer screening’.‘I have more important things to do than go for prostate cancer screening’ and ‘Going through prostate cancer screening would be embarrassing’.The situational barrier mean score was 2.03 (± SD 0.64), indicating only a few barriers perceived among these respondents. Examples of situational barrier items include The ‘contributory factor’ mean score was 2.66 (± SD 0.75), which indicates a moderate perceived risk of getting PCa among these respondents. Examples of contributory factor items include ‘I believe it is likely I will get prostate cancer at some time in the future’ and ‘I think African men are more likely to develop prostate cancer than white men’.The majority of the respondents had multiple sources of knowledge regarding PCa, which included health education from doctors and nurses (52%), media  (50%), literature  (26%), family members (19%) and friends (17%).Most of the respondents (82.8%) felt the need for additional knowledge regarding PCa; aspects requiring additional knowledge were symptoms (78%), risk factors (76%), treatment (66%), the value of PSA (63%), the value of DRE (59%), diagnosis (56%) and the importance of certain nutrients in PCa prevention (48%). Methods suggested by the respondents for the dissemination of additional knowledge included informational leaflets or pamphlets (73%), talking to healthcare providers (e.g. doctors and nurses) (50%), radio (46%), social media (e.g. Facebook and WhatsApp) (46%), home visits by healthcare providers  (45%), television (41%) and posters in public spaces (26%).The ‘screening intent’ mean score was 2.88 (± SD 0.70), reflecting a moderate level of intention to screen for PCa among these respondents. Examples of screening intent items include ‘I intend to have a prostate cancer screening test (prostate-specific antigen blood test) in the next 6 months’ and ‘In the next 6 months, I plan to discuss prostate cancer screening with a doctor’.The relationship between certain background characteristics, cultural factors and beliefs, knowledge and the intention to screen for PCa was assessed using the chi-square test, as shown in p < 0.001), higher perceived benefits of PCa screening (p = 0.002), lower perceived situational barriers to PCa screening (p = 0.006) and higher perceived risk of getting PCa (p = 0.024).The following factors were associated with greater intent to go for PCa screening: lower degree of fear/apprehension of PCa screening . In a similar Kenyan study, the mean age was 49.8 (± SD 16.7) years  were married, 69.7% had at least secondary level education and 35.5% had no income or depended on a government grant. These socio-demographic characteristics seem typical of the study setting, as corroborated by findings of a similar study where 64% of the respondents were married, 53.2% had at least secondary level education and 30% were unemployed  and less than a tenth (6.2%) of the respondents had ever done a PSA test and DRE, respectively, in the past. This may imply a low level of risk perception and the lack of screening opportunities in the community as the respondents included in this study have at least heard about the disease. In the study setting, there may also be the need to reiterate health education on this disease. In a similar study, only 2.4% of the respondents had ever done a PSA test in the past  respondents felt they were at risk of getting PCa, whereas others felt either they were not at risk or were unsure. This may explain the low screening rate found in this study, as explained above. This is unlike a similar study conducted in Kenya, where 64% of the respondents thought they might be at risk of getting the disease  of the respondents preferred spiritual or traditional healers as their first-line healthcare providers. This may be a reflection of the religious and cultural beliefs in the study setting. In a study by Chali, Hasho and Koricha , 81.5% oEven though ‘having previously heard about prostate cancer’ was one of the eligibility criteria for this study, less than one-quarter of the respondents (24.4%) scored ≥ 50%. This is unlike a similar study where a moderate to high level of knowledge was found among 79.1% of those who had previously heard of PCa , literature , family members and friends. This finding is similar to a Kenyan study where respondents’ prior sources of knowledge were radio, health facility and healthcare provider and media   and a high degree of fear/apprehension (mean score of 3.2). They, however, also had strong beliefs in the benefits of screening (mean score of 4.02), and a great influence of family members on screening (mean score of 3.8) , which may have a negative impact on the reliability of the results. However, the results may further contribute to the body of literature on knowledge, cultural factors (and belief) and screening intentions of African men regarding PCa screening, which is presently scarce in South Africa.The respondents in this study have a low level of knowledge and practice of PCa screening. Both fatalistic belief and fear/apprehension towards PCa screening are low. The observed low level of knowledge and practice of PCa screening could be amenable to enhanced educational and awareness strategies, including clarification of cultural misconceptions regarding PCa screening – the respondents’ self-acclaimed knowledge gaps and preferred methods for knowledge delivery should be borne in mind. Factors shown to be associated with greater screening intent should be promoted. Appropriate strategies should also be directed at socially influential people in the lives of these men, including their preferred healthcare providers.Lastly, the study findings should be communicated to PHC providers and other relevant stakeholders. Given the necessary support of the government and policymakers, early diagnosis and prevention of aggressive disease can be enhanced in the community through health education and awareness.


Configurational entropychange is a central constituent of thefree energy change in noncovalent interactions between biomolecules.Due to both experimental and computational limitations, however, theimpact of individual contributions to configurational entropy changeremains underexplored. Here, we develop a novel, fully analyticalframework to dissect the configurational entropy change of bindinginto contributions coming from molecular internal and external degreesof freedom. Importantly, this framework accounts for all coupled anduncoupled contributions in the absence of an external field. We employour parallel implementation of the maximum information spanning treealgorithm to provide a comprehensive numerical analysis of the importanceof the individual contributions to configurational entropy changeon an extensive set of molecular dynamics simulations of protein bindingprocesses. Contrary to commonly accepted assumptions, we show thatdifferent coupling terms contribute significantly to the overall configurationalentropy change. Finally, while the magnitude of individual terms maybe largely unpredictable a priori, the total configurational entropychange can be well approximated by rescaling the sum of uncoupledcontributions from internal degrees of freedom only, providing supportfor NMR-based approaches for configurational entropy change estimation. The fact that thesmallest complex in this study is comprised of 1094 atoms togetherwith the fact that the span of ΔS1D(Y) increases already by a factor of 2 demonstratesthe importance of these external degrees of freedom as well as allof their couplings when investigating small systems. Thus, from anentropic point of view, retaining as much rotational and translationalfreedom as possible at the binding site should turn out especiallybeneficial for small ligands such as many drug compounds. However,due to the enthalpy/entropy compensation,22 one shouldalso consider the impact on the enthalpic component of any practicaloptimization in this direction.It is of interest, especiallyin the context of rational drug design,to assess whether the above results hold if smaller molecules areinvolved. To investigate this, we have analyzed the relationship betweendifferent configurational entropy contributions normalized by thenumber of degrees of freedom , as proposed in ref  + ΔS1D(B) exhibits an excellent linear correlationwith the total entropy change for both the ubiquitin-containing andthe non-ubiquitin-containing complexes  ensemble with an isotropic pressure of 1 barand a constant temperature of 300 K, while system coordinates wereoutput every 1 ps. The pressure and the temperature were controlledusing the Berendsen thermostat and barostat52 with 1.0 and 0.1 ps relaxation parameters, respectively, and a compressibilityof 4.5 × 10–5 bar–1 for thebarostat. Bond lengths were constrained using LINCS.53 The van der Waals interactions were treated using a cutoffof 14 Å. Electrostatic interactions were evaluated using thereaction-field method,54 with a directsum cutoff of 14 Å and relative permittivity of 61. For the complex 1YD8, due to the lackof a separate structure, the ubiquitin binding partner (human GGA3GAT domain) was extracted from the PDB structure of the complex andequilibrated for an additional 500 ns. The PARENT36 program suite, a configurational entropy package in parallelarchitecture, was used for entropy calculations by applying the MISTapproximation.35 For sampling probability densities,50 bins were used in one-dimensional cases and 50 × 50 = 2500in two-dimensional cases.MD simulations were performed as described previously