After ten days, the biomass had increased by 6-10 times, and the dry matter varied from 4.75-7.65%. The highest yield was obtained from ‘Asia red’, while the lowest was from ‘Koregon red’. However, ‘Koregon red’ and ‘Asia red’ had the highest dry matter. ‘Asia red’ was found to have the highest levels of both Chls and vitamin C compared to the other cultivars, while ‘Koregon red’ exhibited the highest levels of total phenolics and flavonoids. Although variations in the levels of individual glucosinolates were observed, there were no significant differences in the total content of glucosinolates among the five cultivars. ‘Asia purple’ had the highest anthocyanin content, while ‘Asia green 2’ had the lowest. The K, Mg, and Na concentrations were significantly highest in ‘Asia green 2’, and the highest Ca was recorded in ‘Asia purple’. Overall, ‘Asia purple’ and ‘Koregon red’ were the best cultivars in terms of nutritional quality among the tested radish microgreens. These cultivars exhibited high levels of dry weight, total phenolics, flavonoids, anthocyanins, essential and total amino acids, and antioxidant activities. Moreover, the implementation of this vertical cultivation method for microgreens, which relies solely on water and seeds known for their tall shoots during the sprouting could hold promise as a sustainable approach. This method can effectively be utilized for cultivar screening and fulfilling the nutritional and functional needs of the population while minimizing the environmental impacts associated with traditional agriculture practices.Growing microgreens on trays without substrate in a vertical multilayered growing unit offers several advantages over traditional agriculture methods. This study investigated the yield performance and nutritional quality of five selections of radish microgreens grown in sprouting trays, without a substrate using only water, in an indoor multilayer cultivation system using artificial light. Various parameters were measured, including fresh weight, dry matter, chlorophyll, minerals, amino acids, phenolics, flavonoids, anthocyanins, vitamin C, glucosinolates, and antioxidant activity with four different  Low incThe practice of sprouting seeds has a long history, especially in Eastern nations, where eating seedlings is a significant part of culinary tradition , and hasAttaining high-quality microgreens requires careful consideration of both species and cultivar selection, as well as an understanding of how plant growth and development influence the nutritional value and phytochemical content within the same crop . Previouin vitro assays.Besides, the increasing global population and the imperative to reduce the carbon footprint of food production are key drivers behind the global shift toward smart agriculture . So, gro22.1The seeds of five radish cultivars having different shoot colorations, namely ‘Asia green 1’, ‘Asia green 2’, ‘Asia red’, ‘Koregon red’, and ‘Asia purple’ were obtained from Asia Seed Co., LTD, Seoul, Korea. These cultivars are commonly distributed in Korea for the production of sprouts. Given the tendency of radish seeds to produce tall shoots, the method technically termed ‘sprouting’ was used for growing radish microgreens. There were significant variations in both germinability and average 1000 seeds weight among the five cultivars (-2s-1 photosynthetic flux density (PPFD) and 6500K cool daylight spectrum) and an 8 h dark period. The temperature was maintained at 20 ± 2°C throughout the entire growth period until the microgreens reached the stage of fully developed cotyledons, just before the emergence of true leaves  using a completely randomized design with three replications. The growing room consists of several growing units arranged with a spacing of 0.5 m between each unit and the wall. Each growing unit is composed 1.0 x 0.5 m shelf with four latyers, capable of accommodating six growing trays per layer. For our study, a total of 15 growing trays were used, with three trays allocated for each cultivar. These trays were arranged on three layers, with one tray from each cultivar on each layer to ensure replication. Initially, the trays were kept in a dark condition for a period of 2 days. Following the initial 2-day period, the sprouts were exposed to light intensity of 2516 lm . The light cycle consisted of a 16 h light period  of freeze-dried microgreens of the tested radish cultivars were submitted to the National Instrumentation Center for Environmental Management (NICEM) Laboratory. Four minerals  were tested following microwave assisted acid digestion standard method 3051  using in2.3Three samples of freeze-dried microgreens of each radish cultivar were subjected to an extraction process using 75% ethanol, ultrasonic extraction for one hour, and room temperature extraction for 24 hours. After filtering the extract with a 0.2 µL filter, an analysis of amino acid content was conducted by Dionex Ultimate 3000 HPLC using the method described by .2.42.4.1Dimethyl sulfoxide (DMSO) chlorophyll extraction procedure was used as described by . One hunV= volume of solvent W= fresh weight of the extracted tissueTotal anthocyanin  content was analyzed using the pH differential method . Freeze-Where A, V, MW, DF, ϵ, and m stands for the difference between the absorbance values at pH 1.0 and pH 4.5 under 520 nm and 700 nm, the total volume of extract (mL), molecular weight of cyanidin-3-glucoside (449.2 g), dilution factor, molar extinction coefficient  and sample quantity (g), respectively.2.4.22PO4, was injected at a volume of 20 µL and flowed at a rate of 1 mL min-1.Three freeze-dried microgreen samples (1 g) of each cultivar were mixed with 10 mL of 5% meta-phosphoric acid and homogenized for 1 min. The homogenized sample was centrifuged , and the liquid layer of extracts was membrane-filtered (0.22 µm) and analyzed by HPLC as described by  using a 2.4.3-1) or standard was mixed with 1 mL of 10% Folin-Ciocalteu’s phenol reagent and 1 mL of 2% sodium carbonate solution. The absorbance at 750 nm was recorded using a microplate reader  after incubation of the samples at ambient temperature for 90 min in the dark. The results were expressed as milligrams of gallic acid equivalents (GAE) per 100 g of sample (mg GAE 100 g-1) after comparison of the measurement to the calibration curve of gallic acid. For total flavonoids analysis, ethanolic extract (1 mg mL-1) of the extract was mixed with 1.5 mL of ethanol, 0.1 mL of 10% aluminum nitrite solution, 0.1 mL of 1-M potassium acetate solution, and 2.8 mL of distilled water. The mixture was stirred and allowed to react for 30 min. Then, the absorbance was measured at 415 nm using a microplate reader . The measurements were compared to a quercetin (QE) calibration curve, and the results were expressed as milligrams of QE per 100 g of sample (mg QE 100 g-1).The total phenolics and flavonoid contents of freeze-dried radish microgreen samples were measured using the methodology implemented previously in our laboratory and described by 2.4.4The extraction of desulfo-GSLs from the lyophilized 200 mg sample was performed according to 2.5Freeze-dried and ground radish microgreen samples were extracted using the methodology described by , which h2.6® 9.1; SAS Institute Inc., Cary, NC, USA) at a significance level of p< 0.05. To further examine the variations between cultivars, Duncan’s multiple range test was conducted. The data normalization was done by median combined with autoscaling for principal component analysis (PCA), heat map, and correlation analysis. These analyses were performed using MetaboAnalyst 5.0 software  to visualize the differences between cultivars.A completely randomized design with three replicates of trays was used to study the differences between cultivars during growing. After harvesting, data were collected for the above parameters with three replicates. The normality of the data was assessed in Excel, and one-way analysis of variance (ANOVA) was performed using SAS statistical software  exhibited lower biomass compared to the cultivars that have lower average 1000 seeds weight   and ‘Asia red’ (7.41%) exhibited statistically the highest dry matter, followed by ‘Asia purple’ (6.69%) and ‘Asia green 2’ (6.14%), while the lowest (4.75%) was recorded from ‘Asia green 1’ Figure 2-1 DW, respectively. The overall results showed that K is the most abundant element in all cultivars of radish microgreens as compared to the other elements.As indicated in 3.2-1 DW, respectively. Glutamine was the most abundant amino acid (above 39%) followed by histidine (above 16%) and asparagine (above 5%) in all the five tested cultivars. From the results of this study, total essential amino acids were higher in ‘Koregon red’ (24.57 g kg-1) and ‘Asia purple’ (24.46 g kg-1) followed by ‘Asia red’ (23.80 g kg-1 DW) (-1) was recorded from ‘Asia red’ followed by ‘Asia green 2’ (0.25 g kg-1) and ‘Koregon red’ (0.20 g kg-1 DW).In this study a total of 20 amino acids were identified in the microgreens of the tested radish cultivars and the relative proportions of amino acids are significantly dependent on the cultivar Table 1.3.33.3.1-1 DW and 5.02-90.65 mg g-1 DW, respectively. ‘Asia red’ was found to have the highest levels of Chl a, Chl b, and total Chls followed by ‘Asia green 1’. On the other hand, ‘Asia purple’ had the highest anthocyanins followed by ‘Koregon red’, while the lowest records in both Chls and anthocyanins were recorded from ‘Asia green 2’ compared to the other tested cultivars.In this study, significant differences among radish cultivars were observed in both Chls and anthocyanins content 5. Total3.3.2-1 DW) followed by ‘Asia green 2’ (186.20 mg 100 g-1 DW), ‘Asia green 1’ (175.42 mg 100 g-1 DW), ‘Asia purple’ (167.04 mg 100 g-1 DW), and ‘Koregon red’ (121.08 mg 100 g-1 DW), respectively  recorded in ‘Koregon red’ and lowest (158.15 mg 100 g-1) in ‘Asia green 2’. Besides, ‘Koregon red’, ‘Asia red’, and ‘Asia purple’ cultivars had statistically similar highest results of total flavonoids , while the lowest (40.94 mg 100 g-1 DW) was obtained from ‘Asia green 2’.As shown in 3.3.4As presented in 3.4-1 sample concentration. With the same trend, the DPPH scavenging capacity varied from 67.06% in ‘Asia green 2’ to 94.88 and 94.59% in ‘Koregon red’ and ‘Asia purple’, respectively, at 10 mg mL-1 sample concentration. In all the four assays, the values were concurrently higher as the sample concentration increased. Similarly, ABTS, FRAP and reducing power exhibited significantly higher values in ‘Asia purple’ and ‘Koregon red’ , and correlation analysis included all the parameters measured on the microgreens of the five radish cultivars to summarize the results presented in 42 and a spacing of 0.5 m2. This implies a yield of 2.63, 2.84, 3.11, 2.16, and 1.97 kg m-2. Among the tested cultivars, ‘Asia red’ demonstrated the highest yield, whereas ‘Koregon red’ exhibited the lowest yield. However, in terms of dry matter content, ‘Koregon red’ and ‘Asia red’ showed the highest levels, regardless of the variations in the average 1000 seed weight. Notably, the fresh weight was inversely related to the average 1000 seed weight, as observed in ‘Koregon red’ and ‘Asia purple’, which could be attributed to the seed density. The stored food reserves within the seed are enough to attain the cotyledon stage for most vegetable microgreens , namely histidine, threonine, valine, methionine, tryptophan, phenylalanine, isoleucine, leucine, and lysine . AdditioVegetables contain a variety of natural bioactive substances, such as vitamins, minerals, antioxidants, and pigments (Chls and carotenoids) . The fun-1) from ‘Aoush’ radish microgreens grown on a mixture of coco pith, perlite, and vermiculite. Similarly, -1 after comparing purple, red and green radish microgreens grown on a mixture of substrate and quartz sand at different light conditions. Notably, the vitamin C content of the five cultivars grown without media in the present study surpassed the contents reported by -1, respectively, from ‘Aoush’ radish microgreens grown on a mixture of coco pith, perlite, and vermiculite. These findings suggest cultivar dependent variation in the contents of vitamin C, total phenolics, and flavonoids in radish microgreens.In agreement with our findings, Brassicaceae family that related to their rich phytochemicals is the main reason for their consumption recommendation. In addition to cancer-protective effects of glucosinolates and isothiocyanates, studies revealed the antioxidant, anti-inflammatory, anti-diabetic, neuroprotective, and cholesterol-lowering effects of Brassicaceae vegetables  in a multilayered growing unit without any substrate. We have seen a four-fold increase in the yield of radish microgreens per square meter with a vertical sprouting growing system as compared to the conventional growing system. ‘Asia red’ had the highest fresh weight while ‘Koregon red’ had the lowest. However, ‘Koregon red’ and ‘Asia red’ had the highest dry matter. Overall, ‘Asia purple’ and ‘Koregon red’ were found to be the best options for obtaining high-quality nutritional content among the radish microgreens tested, as they exhibited high levels of dry weight, total phenolics, flavonoids, anthocyanins, essential and total amino acids, and antioxidant activities with four different assays. Additionally, this method of cultivating microgreens without substrate and utilizing seeds known for their tall shoots during sprouting could serve as a sustainable model for cultivar screening and meeting the nutritional and functional needs of the population. Moreover, this method has several advantages over traditional agriculture methods, such as efficient space utilization, reduced vulnerability to extreme weather and environmental conditions, elimination of the need for pesticides and fertilizers, and the ability to grow them throughout the year due to their short growing cycle. So, a vertical multilayered growing system for microgreens production could be used as one of the solutions to address the fresh vegetables need of the continuously growing world population, because, it is unaffected by adverse weather conditions and provides reliable year-round production with better use of space and minimum water usage without environmental impacts. The findings suggest that supplementing the current growing method with additional studies involving elicitor treatments on the five cultivars could be beneficial in enhancing the quality of radish microgreens. Such treatments have the potential to stimulate the production of phytochemicals (secondary metabolites), thereby increasing the antioxidant activities and resulting in value added radish microgreens.Conceptualization and methodology, ST, MWB, and CJ; data curation, JL, MWB, and K–S.A. software and formal analysis, ST and HRC.; resources, CJ; original draft preparation, ST and MWB; review and editing, JH, supervision, JH, and CJ; project administration, MWB; funding acquisition, CJ. All authors contributed to the article and approved the submitted version.


Compared with “Exclusive”, “Predominantly” had higher insulin of 53% , “Almost exclusive” had lower total and LDL-cholesterol, and “Partial or none” had a lower fat mass index. (5) Conclusions: Our data suggest that children exclusively breastfed at 6 months of age are overall larger at 5 years, with greater stature, higher fat-free mass but similar fat mass, higher HDL-cholesterol and blood pressure, and lower insulin concentrations compared with predominantly breastfed children. Long-term studies of the associations between BF and metabolic health are needed to inform policies.(1) Background: Breastfeeding (BF) has been shown to lower the risk of overweight and cardiometabolic disease later in life. However, evidence from low-income settings remains sparse. We examined the associations of BF status at 6 months with anthropometry, body composition (BC), and cardiometabolic markers at 5 years in Ethiopian children. (2) Methods: Mother–child pairs from the iABC birth cohort were categorised into four BF groups at 6 months: 1. “Exclusive”, 2. “Almost exclusive”, 3. “Predominantly” and 4. “Partial or none”. The associations of BF status with anthropometry, BC, and cardiometabolic markers at 5 years were examined using multiple linear regression analyses in three adjustment models. (3) Results: A total of 306 mother–child pairs were included. Compared with “Exclusive”, the nonexclusive BF practices were associated with a lower BMI, blood pressure, and HDL-cholesterol at 5 years. Compared with “Exclusive”, “Predominantly” and “Almost exclusive” had shorter stature of −1.7 cm  and −1.2 cm  and a lower fat-free mass index of −0.36 kg/m Noncommunicable diseases (NCDs), such as cardiovascular disease (CVD) and type 2 diabetes (T2D), are leading causes of premature death worldwide . NutritiBreastfeeding (BF) is associated with multiple benefits to children ,7 and haBody composition (BC) and other cardiometabolic markers are important already in childhood, as they may predict the future risk of overweight, T2D, and CVD ,20. MostBetween December 2008 and October 2012, the infant Anthropometry and Body Composition (iABC) birth cohort enrolled mothers residing in Jimma town, Ethiopia, and their apparently healthy newborns, born at term (≥37 weeks of pregnancy) with a birth weight ≥ 1500 g ,24. The BF status at 6 months was assessed via questionnaires at the 6-month visit. Infants were assigned to one of four BF status groups: (1) “Exclusive”, (2) “Almost exclusive”, (3) “Predominantly”, and (4) “Partial or none” following previously suggested classifications . DetailsData collection at 5 years has been described elsewhere . BrieflyA 2 mL venous blood sample was drawn from the children after minimum three hours of fasting. The blood samples were collected in tubes without anticoagulants. Blood glucose and glycosylated haemoglobin (HbA1c) concentrations were determined from the fresh whole blood sample. Glucose was determined using the HemoCue Glucose 201 RT system . HbA1c was determined using a DCCT aligned Quo-Test A1c Analyser . Insulin, C-peptide, triglycerides, total cholesterol, and LDL- and HDL-cholesterol were determined from serum. Insulin and C-peptide were determined by module e601 and lipids by module c501 both of the COBAS 6000 analyser .Information on child sex and maternal ethnicity, education, and occupation were collected through questionnaires at the birth visit . GestatiEthical approval was granted from the Jimma University Ethical Review Committee . The study was registered at ISRCTN.com with the number ISRCTN46718296. Measurements were of no harm and of little inconvenience to the mother–child pairs. Written informed consent was obtained from parents or caregivers prior to enrolment.n).All descriptive data are presented as mean ± SD for continuous normally distributed variables and median (Q1–Q3) for non-normally distributed continuous variables. Categorical variables are presented as percentages followed by number of participants ). β indicates change in units of the outcome from one of the non-EBF groups to the reference group “Exclusive”. Non-normally distributed variables, triglycerides, insulin, and C-peptide were log-transformed prior to analysis. Results of log-transformed variables are presented as percentwise change due to back-transformation. Estimates with 95% CI are visualised as forest plots. We ran three different models for each outcome, chosen a priori based on covariates stated above. Model 1 adjusted for sex and age at 5 years. Model 2 additionally adjusted for birth weight, gestational age, and maternal BMI. Model 3 additionally adjusted for IWI and maternal ethnicity, education, and occupation. BP outcomes were additionally adjusted for height in all three models due to correlation between height and BP . Data an2, 104.3 ± 4.4 cm, 3.86 ± 1.06 kg/m2, and 11.15 ± 0.85 kg/m2, respectively , 0.55 kg/m2 , and 0.89 kg/m2  (model 3). No between-group differences were observed for waist circumference.At 5 years, compared with the “Exclusive” group, the “Predominantly” group was associated with a shorter stature of 1.7 cm  (model 3). Compared with “Exclusive”, the “Almost exclusive”, “Predominantly” and “Partial or none” groups were associated with a lower BMI of 0.56 kg/m2  lower FFMI, and children in the “Partial or none” group had a 0.81 kg/m2  lower FMI (model 3).Compared with “Exclusive”, children in the “Predominantly” group had a 0.36 kg/mCompared with “Exclusive”, the “Almost exclusive”, “Predominantly”, and “Partial or none” groups were associated with a lower systolic BP of 4 mmHg , 5 mmHg , and 4 mmHg , respectively (model 3). The “Almost exclusive” and “Predominantly” groups were additionally associated with a lower diastolic BP.Compared with the “Exclusive” group, the “Predominantly” group was associated with 53.34%  higher insulin concentrations (model 3). Moreover, compared with the “Exclusive” group, lower total and LDL-cholesterol of 11.7 mg/dL  and 10.2 mg/dL , respectively, were observed among children in the “Almost exclusive” group (model 3). Lower HDL-cholesterol concentrations of 4.7 mg/dL , 4.6 mg/dL , and 8.7 mg/dL , respectively, were also observed among children in the “Almost exclusive”, “Predominantly”, and “Partial or none” groups compared with children in the “Exclusive” group (model 3). No between-group differences were observed for glucose, HbA1c, C-peptide, or triglycerides.Compared with EBF during the first six months of life, we found non-EBF to be associated with a lower BMI, systolic BP, and HDL-cholesterol at five years of age. Children in the “Predominantly” group were shorter and had a lower FFMI compared with “Exclusive”. The same trend was observed for the “Almost exclusive” group, whereas “Partial or none” compared with “Exclusive” had a lower FMI. Compared with “Exclusive”, lower total and LDL-cholesterol were observed among children in the “Almost exclusive” group, lower diastolic BP was observed among children in the “Almost exclusive” and “Predominantly” groups, and higher insulin was observed among children in the “Predominantly” group.At 5 years, the average BMI in the population was close to growth standards , and theThe observed higher BMI in the “Exclusive” group compared specifically with the “Partial or none” group does not, however, seem to be explained by a higher FFMI in the exclusive group but rather by a lower FMI among children in the “Partial or none” group. As this population’s average FM percentage at birth of 7% was low compared with high-income reference data of 10–15% ,45, a hiConsistent with our findings for cholesterol, a previous literature review found a trend of higher concentrations among exclusively breastfed children the first years of life and suggested that higher cholesterol is partly a result of the higher cholesterol concentrations in breast milk compared with substitutes . It has,Our findings of lower BP among the “Almost exclusive” and “Predominantly” groups compared with “Exclusive” are contrary to proposed mechanisms that BF has a lowering effects on BP . HoweverOur findings of higher insulin among children in the “Predominantly” group compared with “Exclusive” are in agreement with findings from a previous study, which found breastfeeding to be associated with lower insulin concentrations . HoweverThe strengths of this study include long follow-up with a prospective design reducing the risk of recall bias. There was no recall period as to whether breast milk was the main food given, as the questionnaires covered current breastfeeding behaviour at time of visit. However, a recall of up to six months could occur regarding if water or anything else was given, as questionnaires asked about consumption since birth. This as well as the high-quality BC data is especially unique in regard to what has previously been conducted in low-income settings.To limit the influence of confounding factors, which are inevitable in observational studies, we adjusted for variables considered potential confounders. However, we were not able to adjust for maternal smoking, as these data were not collected. As rates of smoking are low in Ethiopia, especially among women, this has likely not influenced our findings . Use of Exclusion of half of the enrolled participants due to not attending relevant visits has decreased power. Yet, we observed significant differences between groups indicating a reasonable power for at least some variables . EspeciaThe iABC cohort is generalisable with other urban Ethiopian populations and other sub-Saharan African urban settings with similar socioeconomic background. The mother-child pairs were restricted to be residing in Jimma Town and recruited from a hospital, where it is more common for urban families to give birth . FurtherOur findings suggest that children exclusively breastfed at 6 months of age have a higher BMI at 5 years compared with children only predominantly breastfed. However, the larger size resulted from higher fat-free mass and greater stature but similar fat mass. The larger size at 5 years associated with EBF may therefore be considered beneficial for building long term metabolic capacity without increasing metabolic load. Moreover, exclusively breastfed children had higher HDL-cholesterol and blood pressure and lower insulin compared with predominantly breastfed children. Apart from a more favourable BC and stature, following the breastfeeding recommendations are associated with multiple other benefits for both the child and the mother, such as improved immunity of the child, which is especially important in low-income settings. Future studies are needed to clarify whether the observed differences in our study will persist and whether new benefits of EBF will appear later in life due to programming effects that may not manifest until beyond childhood.


Bacillus subtilis transforms itself into a dormant cell type called a “spore”. Sporulation initiates with an asymmetric division event, which requires the relocation of the core divisome components FtsA and FtsZ, after which the sigma factor σF is exclusively activated in the smaller daughter cell. Compartment specific activation of σF requires the SpoIIE phosphatase, which displays a biased localization on one side of the asymmetric division septum and associates with the structural protein DivIVA, but the mechanism by which this preferential localization is achieved is unclear. Here, we isolated a variant of DivIVA that indiscriminately activates σF in both daughter cells due to promiscuous localization of SpoIIE, which was corrected by overproduction of FtsA and FtsZ. We propose that a unique feature of the sporulation septum, defined by the cell division machinery, drives the asymmetric localization of DivIVA and SpoIIE to trigger the initiation of the sporulation program.When faced with starvation, the bacterium  Bacillus subtilis is a gram-positive rod-shaped bacterium that normally divides by binary fission when grown in rich medium, resulting in two identical daughter cells that exhibit similar cell fates  (F is achieved by dephosphorylating an anti-anti-sigma factor (SpoIIAA), which sequesters SpoIIAB and thereby liberates σF  , 22. Actrates σF , 24. Therates σF –29. A pur septum –32, therrulation , which arulation , 35, andrulation .F. We first identified a DivIVA variant that did not display forespore-biased localization at the polar septum that triggered the promiscuous activation of σF in both the mother cell and forespore, resulted in a sporulation defect, and caused the thickness of the polar septum to resemble vegetative septa. The mis-localization of this DivIVA variant was corrected by the overproduction of FtsA and FtsZ, which restored septum thickness, forespore-biased SpoIIE localization, and proper σF activation. We propose that a unique architecture of the polar septum, resulting from the overexpression of ftsA and ftsZ at the onset of sporulation, drives the biased localization of DivIVA to establish an intrinsic asymmetry that initiates the cascade of differential transcription that drives the sporulation program.A recent report revealed yet another level of asymmetry at the polar septum . WhereasF is activated specifically in the forespore, which requires a phosphatase, SpoIIE,  that harbored gfp under control of a σF-dependent promoter produced GFP exclusively in the forespore (R94N) resulted in production of GFP in both the mother cell and forespore in ~60% of cells  localized to the cell poles and polar septum  harboring a deletion in racA, which is known to cause a chromosome anchoring defect, produced chromosome-free forespores. In contrast, only 4% of WT cells and 19% of cells expressing divIVAR94N displayed a chromosome anchoring defect  (spoIID/ΔspoIIM) that was arrested before engulfment and thus displayed a flat septum. Further, since SpoIIE is released into the forespore and subsequently recaptured at the polar septum, we used a strain that did not produce the recapturing protein SpoIIQ to ensure that we were not detecting released, then recaptured, SpoIIE-GFP. At the onset of polar septation, WT DivIVA-GFP exhibited a biased localization to the forespore side of the invaginating septum in 72% of cells (n=67) , SpoIIE-GFP also did not asymmetrically localize to the forespore face of the polar septum  , 43. Adds (n=67)   , which ws (n=67) –B”. Conss (n=67) –E”, and s (n=67) –F”. In crescence –D”. Concr septum –H”. The in vitro , 36, we activity , lane 3,R94N and SpoIIE placement at the polar septum. Since the ftsAZ operon is upregulated at the onset of sporulation  displayed preferential localization of SpoIIE-GFP on the forespore  (R94N displayed thicker polar septa of 36 nm (IQR = 40.5 nm) which, although is thinner than ~80 nm vegetative septa, was nonetheless thicker than a WT polar septum. The overproduction of FtsA and FtsZ, though, largely corrected the thick polar septum defect caused by DivIVAR94N and decreased polar septum thickness to 10 nm (IQR = 58.5 nm). In sum, the results indicate that FtsA and FtsZ influence the correct localization of the DivIVA/SpoIIE complex at the polar septum, likely by changing the architecture of the polar septum (which is manifested at one level by thinness of this septum). Additionally, the observation that DivIVAR94N influences polar septum thickness suggests that the DivIVA/SpoIIE complex reciprocally influences the function of FtsA and FtsZ at the polar septum.Khanna ve septa . Since F45.0 nm) . In contB. subtilis at different points of the cell cycle . Presumably, the overexpression of ftsAZ corrects the DivIVAR94N defect by re-establishing a thin polar septum and restores the asymmetric localization of FtsA/FtsZ and DivIVA/SpoIIE on either side of the polar septum. Finally, we propose that any remaining SpoIIE on the mother cell face of the polar septum is degraded by FtsH  or pDG1731 (for insertion into the thrC locus) . For B. subtilis growth curves, one colony on each strain were first inoculated in 2 ml Difco Sporulation Medium . 150 μl of this suspension was then placed in individual wells of a 96-well plate. Cells were grown at 37 °C for 6 h, with shaking, using a microtiter plate reader (Tecan) and the optical density at 600 nm was measured every 15 min. All Escherichia coli strains and plasmids used in this publication are listed in All lis PY79 ; genotypC locus) , 51. TheStrains were grown in DSM for 24 h at 37 °C at 250 rpm and were then subjected to 80 °C for 20 min in a water bath to kill nonsporulating cells and defective spores. Serial dilutions in phosphate buffered saline were plated on LB agar, incubated at 37 °C overnight. Sporulation efficiency was determined by enumerating colony forming units (cfu) per ml and reported relative to cfu obtained for a culture of WT (strain PY79) grown in parallel.−1 FM4–64 membrane dye and/or 2 μg ml−1 DAPI to visualize DNA, as needed. Finally, 3 μl of cell suspension were placed on a glass bottom culture dish (MatTek) and covered with 1 % agar pad made with SM medium. Cells were viewed using a DeltaVision core microscope system equipped with an environmental chamber at 22 °C. Cells images were captured with a Photometrics cool snap HQ2 camera. Eights planes were acquired every 0.2 μm and the data were submitted to deconvolution using SoftWorx software , a phase-only nematic spatial light modulator , a water objective lens  and a piezo z-stage  . In this2 and 60 W/cm2 for 488 nm and 561 nm, respectively. Further details on 3D SIM reconstruction algorithms and associated software have been described (Images were collected over 2 μm (ensuring we imaged the entire thickness of each bacterium) with an axial step size of 0.125 μm. We used an exposure time of 50 ms per phase, resulting in 27 s imaging time for dual-color volume acquisition. The approximate intensity at the sample plane was 80 W/cmescribed . FollowispoIIE or divIVA are derived from pKNT25 and pUT18 and were co-transformed in E. coli BTH101 strain . 100 μl of the diluted culture were placed in a new 96 well plate and lysed by addition of 10 μl of Lysis Buffer (1 mg mL−1 lysozyme in 1X BugBuster buffer (Sigma)) at room temperature for 15 min. 100 μl of Buffer Z  was added and the beta-galactosidase reaction was started by adding 2 mM  ortho-Nitrophenyl-β-galactoside (ONPG). Hydrolysis of ONPG was monitored by measuring A420 every 5 seconds for 30 min using a microplate reader (Tecan). B-galactosidase activity was calculated by measuring the Vmax of the A420 appearance divided by the OD600. Values were then multiplied by 100000, a coefficient that was chosen empirically to approximate Miller units.Plasmids harboring 1 strain . TransfoStrains were induced to sporulate using the resuspension method. At t = 1.5 h cells were harvested by centrifugation and fixed in 4% formaldehyde, 2% glutaraldehyde in 0.1 M cacodylate buffer, post fixed using a 1% osmium tetroxide solution, then dehydrated sequentially in 35%, 50%, 75%, 95% and 100% ethanol followed by 100% propylene oxide. Cells were infiltrated in an equal volume of 100% propylene oxide and epoxy resin overnight and embedded in pure resin the following day. The epoxy resin was cured at 55 °C for 48 h. The cured block was thin-sectioned and stained in uranyl acetate and lead citrate. The sample was imaged with a Hitachi H7600 TEM equipped with a CCD camera . Septum 1


Erwinia amylovora, is one of the most devastating apple diseases. The selection of cultivars of low susceptibility and the study of the genetic mechanisms of the disease play important roles in fire blight management. The susceptibility level to fire blight was evaluated in 102 accessions originating from Asturias, a cider-producing region located in the north of Spain with a wide apple germplasm. Evaluations took place under quarantine conditions using artificial inoculations of grafted plants. The results revealed wide variation in susceptibility responses and low-susceptible cultivars were identified. In addition, 91 cultivars were genotyped using the Affymetrix Axiom® Apple 480 K SNP array to conduct genome-wide association studies (GWAS). A statistically significant signal was detected on chromosome 10 using the multi-locus mixed model (MLMM). Two genes were identified as major putative candidate genes: a TIR-NBS-LRR class disease protein and a protein containing a development and cell death (DCD) domain. The outcomes of this study provide a promising source of information, particularly in the context of cider apples, and set a starting point for future genetic and breeding approaches.Fire blight, caused by  Malus domestica Borkh.) is one of the most widely cultivated tree fruits in temperate regions. Apple trees can be easily propagated and they bear quality fruit, which is considered one of the top functional foods [The domesticated apple  of noldiana ,29,30,31noldiana  and Khannoldiana . A complnoldiana . Furthernoldiana ,36,37,38noldiana  also repIn addition to being the Spanish region with the highest cider production and a large cider-making tradition ,41, AstuE. amylovora among local accessions and to conduct GWAS in order to determine phenotype–genotype associations.Taking this context into account, the goals of this work were to evaluate the susceptibility level against 2,69 = 1.02; p-value = 0.31). Nonetheless, the isolate FB74 was selected for the phenotypic evaluation since it showed the highest mean area under the disease progress curve (AUDPC) value when compared to FB45 and EW07 , international control see ., was rePhenotypic evaluation revealed a wide variation in host responses. Focusing on those cultivars included in the PDO, low susceptibility (BLUP < −4) were detected in varieties such as ‘Carrandona’, ‘Arbeya’, ‘Durona de Tresali’, ‘Xuanina’ and ‘Lin’. At the other end of the distribution (BLUP > 3), highly susceptible cultivars like ‘Durón Encarnado’, ‘San Roqueña’, ‘Limón Montés’, ‘Perico’, ‘Fuentes’, ‘Regona’ or ‘Meana’ were found. On the other hand, ‘Panquerina’, ‘Durón de Arroes’, ‘Solarina’, ‘Repinaldo de Hueso’ and ‘Ernestina’ are examples of cultivars ranked as moderately susceptible (BLUP from −0.5 to 0.5). BLUP values, as well as the number of replicates that were evaluated per accession, are compiled in All the diploid genotypes hybridized , even when the number of subpopulations was two (k = 2). Similarly, the STRUCTURE HARVESTER revealed the highest Delta K value for k = 2. An in-depth study of the population genetic structure discarded strong clustering in the collection.Population structure analyses are plotted in p-value = 9.92 × 10−9; −log10, located on chromosome 10 position 32,827,282 of the ‘Golden Delicious’ doubled-haploid genome (GDDH13) v. 1.1. Even though no markers exceeded the significance threshold of 6.6 using the mixed linear model (MLM), a convincing peak was identified in the same window region as MLMM. Indeed, the −log10 of AX-115639581 in MLM was 5.9. For both MLM and MLMM, the Q-Q plot indicated a good fit of the model, as shown in A significant signal was detected using the multi-locus mixed model (MLMM) at the bottom of chromosome 10 . The sigR2) by AX-115639581 were 3.22 and 55.61%, respectively. p-value < 0.001), although a wide phenotypic variability can be observed within the heterozygous and homozygous CC groups ; and MD10G1232400, annotated as TIR-NBS-LRR class disease protein. Both are located downstream of the marker, 12 Kbp and 29 Kbp, respectively [R-genes) [E. amylovora [Chromosome 10 has been associated with fire blight resistance in previous studies. In particular, Thapa et al.  identifiu10 gene ,30,68 agectively . TIR-NBSR-genes) . Overexpmylovora ,71,72. Omylovora . It seemmylovora .Since the MLM emerged , MLM-basDeciphering the genomic basis of quantitative variation is crucial to understanding evolution and accelerating plant breeding . AlthougThe identification of a genomic region associated with fire blight can be used to perform a marker-selection approach, which, in turn, can decrease both the time and money required for developing and selecting desirable genotypes . MoreoveIn conclusion, the findings of this study not only offer promising information but also serve as a valuable resource for the selection of optimal cultivars for apple production and for their integration into breeding programs, especially within cider apple growing.Susceptibility was evaluated on potted plants in 103 accessions preserved in the Apple Germplasm Bank of Asturias . Of them−2·s−1), when they continued to grow until they were moved to the high-biocontainment box (BSL3) of the IRTA—CReSA .Phenotypic evaluation was performed by inoculation of growing shoots on potted plants over two years: 2019 and 2021. For each year, eight 1-year-old shoots per accession were whip-grafted onto ‘M7’ EMLA apple rootstock and potted on 770 mL containers with substrate Exclusive . Dormant plants were placed in a cold greenhouse until they reached a length of 5 cm. Then, they were transferred to a cold chamber set at 14 °C and 16/8 h light/dark photoperiod   and watered every two days with tap water at soil level, avoiding shoot or leaf watering.Plants that failed to achieve a growth of 10 cm were not moved to the high-biocontainment box and they were not evaluated. Moreover, the maximum number of replicates per accession and year was set at six and the minimum number at three, considering those accessions with fewer than three valid replicates unsuccessful for that year. Plant material was inoculated one week after moving to the high-biocontainment box, where culture conditions were set at 23 °C, high relative humidity (≈100%) and 16/8 h photoperiod with 300 mmol·mon (PAR) . Plants All plant material was evaluated in a single batch in 2019, and it was divided into two batches due to different growth rates in 2021. Plant material was randomly distributed by blocks in the three batches.E. amylovora by nucleic acid analysis of the 1100 pb rDNA 16S fragment at the Laboratory of Instrumental Techniques of the University of León. FB74 and FB45 were isolated from an infected apple orchard in Catalonia (Spain) and stored at the IRTA—CreSA. EW07 was provided by the Department of Engineering and Agricultural Science .Pathogenicity was performed on three different isolates identified as Eight apple cultivars, ‘Florina’ and seven local ones, were used to test the pathogenicity of the isolates. The local cultivars ‘Collaos’, ‘De la Riega’, ‘Limón Montés’, ‘Meana’, ‘Regona’, ‘Solarina’ and ‘Xuanina’ were selected because they previously showed different levels of susceptibility against the bacteria  and ‘FloThe level of pathogenicity was assessed following the same protocol described above for culture conditions and the same workflow described below for inoculum preparation, inoculation and evaluation.Isolate FB74 stored at −80 °C was sown using the three-phase streaking pattern (T-streak) in fresh LB media and incubated at 25 °C for 48 h. A suspension of 108 cfu/mL was prepared by resuspending the isolated colonies in sterile water. Adjustment was performed using a viable absorbance relationship previously prepared (absorbance = 0.1 at λ = 620 nm) . The inoInoculation was performed by cutting the first completely unfolded leaf using scissors previously immersed in the bacterial suspension . Two cutSusceptibility was recorded 7, 14 and 21 days post-inoculation by dividing necrosis progression by shoot length. Necrosis progression was measured using the scale described by Calenge et al.  Figure : (i) 0—n® Apple 480 K SNP array [To generate the genotypic data, the genomic DNA of 91 diploid accessions  from 1 to 6. After running seven replicates for each k, STRUCTURE HARVESTER v. A.2 [k.The population structure was studied using both PCA and Bayesian clustering. PCA was calculated using the PLINK software, which was also used to prune the set of SNPs using the “indep” option. Pruning was performed by setting a window size of 1 Mbp, a step size of 10 and linkage disequilibrium (LD) measured as E v. 2.2 , settingR v. A.2  was usedSusceptibility data recorded at 7, 14 and 21 days post inoculation were used to calculate AUDPC values in the ‘agricolae’ package v. 1.3-6 . Pathogelme4’ package v. 1.1-34 [lme4’ package.Phenotypic results were adjusted by fitting a mixed linear model using the ‘. 1.1-34 . “AccessGAPIT’ package v. 3.1.0 [10 threshold for significant associations was set to 6.6  at a significant level of 5%. Genes within windows of 100 Kbp in both directions of a significant marker were explored using the JBrowser tool hosted on the Genome Database for Rosaceae (GDR) website [Phenotype–genotype associations were conducted using the MLM and MLMM methods implemented in the ‘v. 3.1.0 , settingv. 3.1.0  using thv. 3.1.0 . After a website .ggplot2’ package v. 3.4.2 [All analyses were implemented in the R software environment v. 4.3.1  and the v. 3.4.2  was used


The increasing incidence of pregnancy complications, particularly gestational diabetes mellitus (GDM) and preeclampsia (PE), is a cause for concern, as they can result in serious health consequences for both mothers and infants. The pathogenesis of these complications is still not fully understood, although it is known that the pathologic placenta plays a crucial role. Studies have shown that PPARγ, a transcription factor involved in glucose and lipid metabolism, may have a critical role in the etiology of these complications. While PPARγ agonists are FDA-approved drugs for Type 2 Diabetes Mellitus, their safety during pregnancy is not yet established. Nevertheless, there is growing evidence for the therapeutic potential of PPARγ in the treatment of PE using mouse models and in cell cultures. This review aims to summarize the current understanding of the mechanism of PPARγ in placental pathophysiology and to explore the possibility of using PPARγ ligands as a treatment option for pregnancy complications. Overall, this topic is of great significance for improving maternal and fetal health outcomes and warrants further investigation. Pregnancy can lead to complications that pose serious risks to both the mother and infant during pregnancy, labor, and postpartum. These complications typically arise from conditions unique to pregnancy. Alarmingly, there has been a 16.4% increase in the incidence of pregnancy complications between 2014 and 2018, with gestational diabetes mellitus (GDM) increasing by 16.6% and preeclampsia (PE) increasing by 19% ,4,5. WomTo examine the role of PPARγ in GDM and PE, with a particular focus on placental pathophysiology, this review conducted a comprehensive search using the PubMed database. Only original research and scientific abstracts published between 2005 and 2022 that investigated the role of PPARγ in GDM and PE were included. The search terms used were “peroxisome proliferator-activated receptor-gamma”, “PPARγ”, “PPAR gamma”, “gestational diabetes mellitus” and “preeclampsia”. Studies involving other PPARs and other pregnancy complications such as infertility, hypertensive pregnancy, and polycystic ovarian syndrome were excluded .The Peroxisome Proliferator-Activated Receptor-γ (PPARγ) is a PPAR subfamily member consisting of two isoforms. PPARγ1 is encoded by mRNA PPARγ1, PPARγ3, and PPARγ4, while PPARγ2 is translated from mRNA PPARγ2 . PPARγ1 PPARγ is a ligand-dependent transcription factor, meaning it can be regulated by agonists and antagonists. It acts as a sensor for different fatty acid types, also known as a lipid sensor. In addition to endogenous ligands, synthetic ligands are widely used in clinical practice and in vitro studies to modulate PPARγ ,14. A suPPARγ, in addition to its well-established roles in lipid metabolism and adipocyte differentiation, has also been shown to be essential in regulating insulin resistance, glucose metabolism, immunity, as well as cell biology, including cell differentiation ,46,47. TPPARγ is highly expressed in human placentas, particularly in syncytiotrophoblasts, cytotrophoblasts, and extravillous trophoblasts (EVTs) ,50. Its Recent in vitro studies have shown that PPARγ is associated with trophoblast migration and invasion, although its exact role in these processes appears to be paradoxical. Some studies have reported that PPARγ inhibits trophoblast invasion in human primary cultures of EVTs ,53,54. OHowever, more recent studies have suggested that PPARγ may promote trophoblast migration. Activated PPARγ/RXRα heterodimer by IL-17 was found to promote proliferation, migration, and invasion in HTR8/SVneo, a trophoblast cell line . FurtherPPARγ has also been identified as a regulator of trophoblast differentiation. In the BeWo cell model, blocking PPARγ activity has been shown to induce cell proliferation but suppress the differentiation . In humaPPARγ single nucleotide polymorphisms (SNPs) are associated with increased susceptibility to pregnancy-related diseases, including GDM and PE. The rs201018 and C1431T variants of PPARγ have been reported to be significantly associated with susceptibility to PE in different populations ,55, withIn addition to PE, PPARγ SNPs have also been associated with the incidence of GDM. The Pro12Ala polymorphism of PPARγ is one of the dominant variants associated with GDM susceptibility, as reported in several meta-analyses ,57,58. HPE is a serious pregnancy complication that affects 5–7% of pregnancies worldwide  and is rPE can have serious consequences for both the mother and the baby. If the placenta is not implanted correctly, it can result in inadequate blood flow to the placenta, leading to fetal growth restriction or intrauterine growth restriction (IUGR) . This caDiagnosis of PE typically involves measuring blood pressure, with systolic blood pressure greater than 140 mmHg and diastolic blood pressure greater than 90 mmHg, along with proteinuria . PE is uThe role of PPARγ in the pathogenesis of PE is still not fully understood, but studies have shown conflicting results regarding its expression levels in PE placentas. While some studies have reported increased expression of PPARγ in PE placentas ,76, mostOne mechanism that has been explored is the modulation of 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), a gene that helps in the maintenance of cortisol levels in the placenta. PPARγ was found to positively correlate with 11β-HSD2 expression in PE placentas, and treatment with rosiglitazone, a PPARγ agonist, increased the expression of 11β-HSD2 in placental explants, while treatment with GW9662, a PPARγ antagonist, decreased the expression of 11β-HSD2. Interestingly, the effect of PPARγ ligands was blocked when specificity protein 1 (Sp-1) was knocked out . AnotherPPARγ has also been shown to participate in regulating sFlt-1 secretion through nuclear factor erythroid 2-related factor 2 (Nrf2) . ProcyanIn addition, studies have shown that PPARγ and angiopoietin-like protein 4 (ANGPTL4), which is associated with fat metabolism and vessel formation, are reduced in PE placentas compared with control placentas . TreatmePPARγ has been implicated in regulating epigenetic modifications in the placenta, specifically histone methylation and acetylation. Epigenetic modifications play a crucial role in regulating gene expression and can impact various cellular processes, including trophoblast invasion and migration . The co-Overall, the role of PPARγ in the pathogenesis of PE is complex and still not fully understood, with conflicting findings in different studies. Further research is needed to elucidate the exact mechanisms and potential therapeutic implications of PPARγ in PE.Although human placental tissue and explants can provide valuable information about the function of PPARγ in PE, ethical concerns make it impossible to determine the effect of its ligands in human studies. Therefore, rodent models, such as mice and rats, have been widely used to study the molecular mechanisms of PE. One commonly used rodent model is the reduced uterine perfusion pressure (RUPP) model in pregnant rats, which involves creating an abdominal incision on E14.5 of gestation to mimic abnormal uteroplacental blood flow . RUPP raOn the other hand, treatment with T0070907, an antagonist of PPARγ, in mice has been shown to induce hallmark symptoms of PE, including hypertension, proteinuria, and fetal growth restriction . These mBoth animal models and human tissue studies have shown a decrease in PPARγ expression in the pathogenesis of induced or diagnosed PE, respectively. This suggests that PPARγ could be targeted by pharmacological interventions to potentially reduce the severity of PE in pregnant women diagnosed with the disease.Potential treatments for PE can target PPARγ or genes associated with PPARγ, leading to significant changes in PPARγ expression that positively impact women or result in mice displaying hallmark signs of the disease. Treatment with the PPARγ antagonist T0070907 induced PE-like symptoms in mice . HoweverGestational diabetes mellitus (GDM), a form of glucose intolerance that arises during pregnancy, affects approximately 7.6% of pregnancies in the US and is one of the most common obstetric complications . AccordiAs the intermediate transportation site between mother and fetus, placentas from GDM also displayed a pathophysiological change in the spiral artery and vasculature . While tPPARγ agonists, such as rosiglitazone, are commonly used drugs to treat T2DM . HoweverThe expression changes of PPARγ in the placentas of patients with GDM have yielded inconsistent results across various studies. While some studies have reported downregulation of placental PPARγ, This can result in placental ischemia, or reduced blood flow to the placenta, due to failed remodeling of spiral arteries and impaired vascularization expression in GDM patients compared with healthy control groups ,114,115,In vitro studies have demonstrated that hyperglycemic conditions can impair placental vascularity through the repression of migration and viability . PPARγ hCurrently, there is substantial evidence supporting the critical role of PPARγ in placental biology. PPARγ is highly expressed in trophoblasts, and its expression is influenced by maternal factors. While numerous in vitro studies have demonstrated that PPARγ regulates trophoblast differentiation and migration, there is still limited in vivo information that provides a comprehensive understanding of placental physiology and the complex pathophysiology of pregnancy. Several genes and chemicals have been identified to interact with PPARγ in trophoblasts, including COX, 11β-HSD2, angiotensin 1–7, HO-1, GCM1, Nrf2, ANGPTL4, CTRP6, DsbA-L, and aspirin. These interactions offer potential mechanistic insights into the function of PPARγ in the placenta. However, the current conclusion of PPARγ in placentas affected by GDM remains inconsistent. Determining whether PPARγ plays a protective role in the progression of GDM requires further in vivo studies, as systematic hyperglycemia may differ from that observed in in vitro models.Furthermore, the ability of PPARγ ligands to modulate trophoblast function underscores the promising potential of PPARγ as a therapeutic target in the treatment of conditions. However, as depicted in


Cancer-related fatigue (CRF) is an extremely common and long-term condition that affects the physical and mental health of oncology patients. While the treatment for CRF with western medicine and non-pharmacological therapy remains uncertain and challenging, traditional Chinese medicine (TCM) has become a trending option for the patients. Based on the findings from randomized controlled trials (RCTs), this study aims to identify and evaluate the evidence about the efficacy and safety of TCM for CRF.A systematic literature search was conducted according to the PRISMA literature research guidelines. Seven electronic databases including PubMed, the Cochrane Library, Embase, Web of Science, Scopus, China National Knowledge Infrastructure (CNKI) and Wanfang database were searched to identify RCTs which investigated TCM in the treatment of CRF published since inception to December 2022. RCTs comparing TCM with no treatment, placebo, or pharmacological interventions were considered eligible for this review. The Consolidated Standards of Reporting Trials Statement extensions for Chinese herbal medicine Formulas (CONSORT-CHM) and the Cochrane Collaboration’s Risk of Bias tool were used in this review to evaluate the quality and the risk of bias of all included trials.Astragali Radix, Ginseng Radix and Codonopsis Radix. Some TCM products, such as Kangai Injection, Buzhong Yiqi Decoction and Shenqi Fuzheng Injection could provide a reference for medication in this review. A range of non-serious, reversible adverse effects associated with the use of TCM was also reported. However, the result of evaluation showed that none of the trials fully met all the CONSORT-CHM criteria, the quality of included trials was generally poor and the risk of bias was mostly uncertain.A total of 82 RCTs were included in this review, regardless of whether they were published in English or Chinese. After data extraction and results evaluation, 78 trials demonstrated overall efficacy in using TCM for CRF patients compared with the control group, in which 33 trials showed that the efficacy rate was statistically significant (p < 0.05 or p < 0.01). TCM was also shown to be beneficial in improving the scores of relevant scales  or physical tests indicators . The most common herbs found in Chinese medicine were There is some evidence supporting the efficacy and safety of TCM in managing CRF in this systematic review. However, no clear conclusion can be made due to the inadequate reporting of efficacy and adverse reactions. In view of some concerns about the existing evidence after the evaluation, it is essential to standardize the comprehensive identification and efficacy measurement standards, improve the quality of RCTs and conduct more multicomponent therapies to provide an updated reference for CRF patients medication in the future.https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42023413625].The protocol of this systematic review has been registered on PROSPERO (CRD42023413625). [The online version contains supplementary material available at 10.1186/s13020-023-00849-y. Cancer-related fatigue (CRF) refers to a persistent, subjective sense of tiredness or exhaustion related to cancer or cancer treatment that is not proportional to recent activity and interferes with usual functioning . UnderlyCurrently, there is not a standard approach to CRF treatment. Western medicine therapies and non-pharmacological therapies have been commonly employed to help alleviate the fatigue experienced by oncology patients , 5. The qi and blood deficiency, disharmony of yin and yang, hypofunction of liver, kidney, spleen or other organs .This study was a systematic literature review conducted and reported in compliance with the updated Preferred Reporting Items for Systematic Reviews and Meta analyses (PRISMA) guidelines . The ConRandomized controlled trials which investigated the use of TCM in CRF regardless of blinding, status, date or language of publication were considered eligible for this review. Open-label experiment and observational studies were excluded.Participants of any age, gender or ethnic origin with a diagnosis of CRF, or the presence of fatigue after chemotherapy were eligible for the studies. RCTs in which participants had other diseases resulting in fatigue symptoms, or non-drugs treatments were excluded.The interventions in the included RCTs for the treatment of CRF were divided into two categories, primary interventions and behavioral interventions. In particular, the primary interventions usually included TCM, western medicine (WM), TCM plus western medicine (TCM + WM), placebo or no treatment; behavioral interventions were set for chemotherapy (CH), other treatment (OT) and chemotherapy plus other treatment (CH + OT). Other treatment (OT) referred to the conventional treatment, symptomatic treatment, supportive treatment, nutritional therapy or other non-drug treatment for cancer patients after chemotherapy.TCMWestern medicine (WM)TCM plus western medicine (TCM + WM)PlaceboNo treatmentPrimary interventions:Chemotherapy (CH)Other treatment (OT)Chemotherapy plus other treatment (CH + OT)Behavioral interventions:Both efficacy and safety of the TCM investigated in the included RCTs were analyzed. As such, the primary and the secondary outcomes of managing CRF with the use of TCM were as follows:The primary outcome measures under consideration included changes in Piper Fatigue Scale (PFS) and the scales of QoL. On the one hand, PFS consists of twenty-two items and four subscales in total, six items of behavioral/severity, five items of affective meaning, five items of sensory and six items of cognitive/mood , 20. Eac1 ; QoL2  for cancer patients (Chinese version draft) were used to evaluate the QoL when TCM was used for treating CRF in this review.On the other hand, QoL is defined as: “an individual's perception of their position in life in the context of the culture and value systems in which they live and in relation to their goals, expectations, standards and concerns” from the World Health Organization (WHO) , 22. As TCM syndrome score: According to the Chinese Medicine Clinical Research of New Drugs Guiding Principles , TCM synFatigue scales: BFI (Brief Fatigue Inventory), CFS , CFS* , MFSI-SF , FSI (Fatigue Symptom Inventory), BFI-C (Chinese Version of Brief Fatigue Inventory), TOI-F , VAS-F , NGFRS Other scales: HADS , POMS (Profile of Mood States), HAMA , SDS , SS (satisfaction survey), CS (Comfort Survey), PSS , GIC , LASA , BDI , GCSG , LCSG +, CD4+, CD8+, CD3+CD4+, CD3+CD8+, CD4+/CD8+, CD16+56+, TNF-α, IFN-γ, IL-1, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, NK cell, IgG, IgA, IgM, TGF-β.Cytokines tests: CD3Blood tests: hemoglobin (HGB), albumin (Alb), white blood cell (WBC), total protein (TP), platelet (PLT), cortisol (COR), red blood cells (RBC), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), hematocrit (HCT), degree of myelosuppression (DM)Others: weight, cardiac function, liver function, kidney function, blood viscosity, urinalysis, Recombinant Human Granulocyte Colony Stimulating Factor (rhG-CSF) dosage, physical and chemical indicators, WHO Response Evaluation Criteria in Solid Tumors (RECIST), improve cancer cachexia-related symptoms, standards for the diagnosis and treatment of common malignant tumors in China.The secondary outcome measures were TCM syndrome score, fatigue scales, cytokines tests, blood tests, other scales and indicators:This systematic review was conducted according to the PRISMA literature research guidelines . Seven eAs shown in Table This review only included studies that reported the results of RCTs investigating the efficacy and/or safety of TCM in CRF. Studies subjected to exclusion were: (1) review, meta-analysis, protocol; (2) non-randomized trial; (3) pharmacodynamics or pharmacology studies; (4) animal experiments; (5) studies on other non-drugs therapy ; (6) studies on other diseases or fatigue caused by other diseases.All references were categorized and archived in Endnote X9, data was recorded and organized using the EXCEL 2013. As listed in Table Two of the authors  independently assessed the included eligible studies based on 25-item of CONSORT-CHM 2017 statement [Cochrane Collaboration’s Risk of Bias  tool wasThe screening process conducted in accordance with the PRISMA guidelines is summarized in the flow diagram as shown in Fig. Since one of the included studies reported two RCTs , this syA total of 7547 participants with CRF were recruited in the RCTs included in this review. After 312 participants dropped out or withdrew before the intervention was initiated, the remaining participants in 82 trials were allocated into the test groups n = 3801) and the control or comparison groups n = 3434). Excluding eight trials which did not report the number of males and females [01 and th4. ExcludThe top three categories of cancer in 82 trials were lung , bowel  and breast , followed by stomach, liver, ovarian, cervical, esophageal, kidney, ureteral, pancreatic, gallbladder, nasopharyngeal carcinoma, head and neck or other categories. Regarding places of recruitment, 73 trials recruited participants from hospitals, one from newspaper or hospital advertisements , one froInformation about the diagnosis and inclusion criteria of the CRF participants in this review is provided in Additional file Other scales such as PFS, KPS, ECOG, BFI, QLQ-C30, CFS*, NGFRS, VAS, a 11-point fatigue scale and a question about fatigue level were used as the tool to determine hte inclusion criteria for CRF participants in 52 trials. In particular, the score of KPS was set between 40 to 80 in 34 trials included in this review. Seven trials used ECOG with the score less than two. Seventy-two trials used tumor diagnosis and 10 trials had no information regarding the tumor diagnosis. One trial mentioned the level of HGB > 80 g /L , one triIn terms of life expectancy, 31 trials did not provide any information whereas 51 trials discussed different life expectancy periods, with more than 30 days in one trial, more 1–3 months in 33 trials, more than 4 months in one trial, more than or equal to 6 months in 15 trials and more than 9 months in one trial.Kangai Injection (n = 16) (康艾注射液), Buzhong Yiqi Decoction (n = 9) (補中益氣湯), Shenqi Fuzheng Injection (n = 6) (參芪扶正注射液), Guarana (Paullinia cupana) (n = 4) and Shenfu Injection (n = 3) (參附注射液).All the ingredients from each TCM preparation investigated in the included studies are summarized in Table Astragali Radix(黃芪) (n = 48), Ginseng Radix(人參) (n = 34), Codonopsis Radix(黨參) (n = 30), Atractylodis Macrocephalae Rhizoma(白朮) (n = 30), Glycyrrhizae Radix(甘草) (n = 25), Poria (茯苓) (n = 21), Angelicae Sinensis Radix (當歸) (n = 21), Sophorae Flavescentis Radix (苦參) (n = 17), Citri Reticulatae Pericarpium (陳皮) (n = 15), Bupleuri Radi (柴胡) (n = 12), Cimicifugae Rhizoma (升麻) (n = 11), Ophiopogonis Radix (麥冬) (n = 8), Rehmanniae Radix (地黃) (n = 8), Dioscoreae Rhizoma (山藥) (n = 7), Coicis Semen (薏苡仁) (n = 7), Lycii Fructus (枸杞) (n = 7), Ziziphus Jujuba Mill (大棗) (n = 6), Paeoniae Radix Alba (白芍) (n = 6), Pinelliae Rhizoma (半夏) (n = 6) and Amomi Fructus (砂仁) (n = 5).At least 100 Chinese medicinal herbs were involved in the included trials for treating CRF and the 20 most common herbs were In test group, the primary interventions were designed for TCM (n = 80) and TCM plus western medicine (n = 2). Thirty-seven trials used other treatment, 15 trials considered chemotherapy and 17 trials provided chemotherapy combined with other treatment as behavioral interventions. The primary interventions in control groups included placebo (n = 10), TCM (n = 2), western medicine (n = 3) and no treatment (n = 1). Thirty-nine trials used other treatment, 17 trials considered chemotherapy and 17 trials provided chemotherapy combined with other treatment as behavioral interventions. All the comparisons were described in Table The duration of the intervention ranged from 10 days to 16 weeks. The intervention durations were shorter than or equal to 1 month in 59 trials, between 1–2 months in 12 trials, between 2–3 months in 7 trials, more than 3 months in one trial, two cycles of treatment in 1 trial and four cycles of treatment in 1 trial. One trial did not provide any information regarding intervention duration.As shown in Table PFS was considered a measuring tool for CRF and was selected by more than half of the trials (n = 46).In addition, scales of QoL were selected in 51 trials, and 9 trials employed more than two scales. The most common scales included KPS (n = 27) and QLQ-C30 (n = 17), other were PSQI (n = 3), SF-36 (n = 3), FACIT-F (n = 3), GQoLI-74 (n = 2), FACT-F (n = 2), FACT-G (n = 1), FACT-ES (n = 1), Quality of Life Questionnaire (n = 2), but 31 trials did not provide any information.Twenty-seven trials selected TCM syndrome score.Fatigue scales included BFI or BFI-C (n = 17), CFS (n = 5), BFI combine CFS (n = 1), BFI combine MFSI-SF (n = 1), BFI combine CFS* (n = 4), FSI (n = 3), FSI combine MFSI-SF (n = 1), NCCN GFRS (n = 1), VAS-F combine TOI-F (n = 1).Other less commonly used scales were HADS (n = 5), GIC combine LASA (n = 1) and HADS combine SDS (n = 1). The scales of BDI, CS, GCSG, LCSG, POMS, PSS, SS were only used once.+, CD4+, CD8+, CD3+CD4+, CD3+CD8+, CD4+/CD8+, CD16+56+, TNF-α, IFN-γ, IL-1, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, NK cell, IgG, IgA, IgM and TGF-β.For the cytokines test, 15 trials reported the results of CD3Out of the 11 trials that reported the results of blood test, 5 trials conducted HGB test, WBC combined PLT were reported in three trials, COR and DM were separately reported in two trials, and the remaining parameters including Alb, TP, RBC, MCH, MCHC, MCV, HCT were only reported once. Seventy-one trials did not provide any information regarding blood test results.Other outcomes included RECIST (n = 5), weight (n = 2), cardiac function (n = 2), liver function (n = 2), kidney function (n = 2), rhG-CSF dosage (n = 2), blood viscosity (n = 1), urinalysis (n = 1), physical and chemical indicators (n = 1), improvement in cancer cachexia-related symptoms (n = 1), standards for the diagnosis and treatment of common malignant tumors in China (n = 1).The results of the included trials showed that 78 trials reported an overall efficacy on the use of TCM in treating CRF whereas only 4 trials showed no effect.Out of the 34 trials which demonstrated the efficacy rate in test groups as compared to control groups, 28 trials showed statistically significant difference (p < 0.05), 5 trials showed statistical differences at a two-sided P-value less than 0.01, while one trial showed the negative difference (p > 0.05). In particular, the efficacy rate in test groups ranged from 15.9 to 100%, 11 trials were more than 90%, 20 trials were over 50% and less than 90% and 3 trials were below 50%. The efficacy rate in control groups ranged from 6.96 to 80% with 15 trials over 90% and 19 trials below 50%. Based on the different classification of TCM dosage forms, the efficacy rate of test groups considering TCM injections ranged from 15.9 to 92.31% while for TCM decoctions in 15 trials, the range was 70.00 to 100.00%.Kangai Injection (n = 16), Buzhong Yiqi Decoction (n = 9) and Shenqi Fuzheng Injection (n = 6), all show overall efficacy in this review. In 16 trials, Kangai Injection not only effectively improved the scores of PFS, BFI, FSI, QLQ, HAMA, SDS and other scales, but also improved the level of IL-1β [The three most used TCM products, such as of IL-1β , 92–96. of IL-1β , 82, 84,of IL-1β , 94–96, of IL-1β , 92, 93 of IL-1β . A totalBuzhong Yiqi Decoction was not only used in China [Bojungikki-Tang in traditional Korean medicine [Among 9 included RCTs, in China , 76, 91,medicine , and themedicine , 76.Shenqi Fuzheng Injection [In 6 included trials studying njection , 70–72, njection , 60, 71.Zini Yiqiyangxue Recipe (自擬益氣養血方) was fully effective in treating patients with colorectal cancer fatigue, with the overall efficacy rate of 100% in the test group as compared to 48% in control group (p < 0.01). After the intervention, it was reported that 9 cases were obviously effective and 16 cases were effective in the test treatment group, and nearly half of the control group were ineffective (13/25) [One trial found that  (13/25) .American ginseng (Panax quinquefolius) and Guarana (Paullinia cupana) should be carefully considered in this review. Two trials that considered American ginseng as a TCM intervention had mixed results in terms of overall efficacy [American ginseng alone improved fatigue and QoL [American ginseng with other treatments did not significantly improve BFI scores [Guarana, only 1 of 4 trials in this review supported its use for fatigue relief [Paullinia cupana (PC-18). One trial compared PC-18  with placebo, while another trial compared either 7.5 mg or 12.5 mg of PC-18 with placebo, and the results of both trials showed no statistical difference between the two groups [Guarana group  and placebo with the scores of CFS*, BFI or BDI [Guarana  significantly improved FACIT-F, FACT-ES, and BFI scores compared to placebo on day 21 and day 49 (p < 0.01) [The efficacy of efficacy , 32. One and QoL , but thee relief , and thee relief , 26. In o groups . Similar < 0.01) .Among the 82 trials included, only 3 trials reported no adverse reactions , 52, 74,Korean red ginseng (KRG) [American ginseng [Paullinia cupana [Wisconsin Ginseng [Only 5 trials reported several serious adverse reactions (Common Terminology Criteria for Adverse Events level ≥ 3) during TCM treatment in the test group , 33, 96. ginseng , Paullina cupana  and Wisc Ginseng  were useThe summary of the CONSORT-CHM quality assessment results of the 82 RCTs is shown in Additional file The six domains of the risks of bias evaluation for included trials are shown in the following and the summary of the risk of bias assessment is shown in Fig. Twenty-six trials were at a low risk of bias due to the application of random sequence generation, 9 trials were deemed having a high risk of bias based on admission order, hospital number, medical record number or other order , 95, 103Four trials reported detailed information were regarded as low risk of bias , 33, 34,Five trials reported using double-blinded design were regarded as low risk , 32–34 aThree articles mentioned blinding of outcome measurers , 33, 34.All trials did not provide detailed information.Since results of some scale points were not presented in a trial, it was regarded as having high risk of bias. The remaining 81 trials were evaluated at an unclear risk of bias.There was no baseline population characteristics in a trial . One triAstragali Radix (黃芪), Ginseng Radix (人參) and Codonopsis Radix (黨參)) or finished products (including Kangai Injection (康艾注射液), Buzhong Yiqi Decoction (補中益氣湯) and Shenqi Fuzheng Injection (參芪扶正注射液)). The benefits of TCM were reported in terms of improvements in the scores of the fatigue scales as well as physical indicators . Overall, the findings of this review suggested that some TCM were effective in the management of CRF and relatively safe to use. However, the positive findings of the current review should be interpreted with cautions due to the concerns about the quality of the included RCTs. The implications of the review findings about TCM for CRF on clinical practice and the methodology of RCTs will be discussed in further details in the following.This systematic literature review included 82 RCTs which assessed the efficacy and safety of TCM for the treatment of CRF. Regarding efficacy, 78 studies reported an overall efficacy of TCM in treating CRF compared with the control group regardless of whether behavioral interventions were involved, in which 33 trials demonstrated that the efficacy rate was statistically significant (p < 0.05 or p < 0.01). Regarding the safety of TCM under investigation, non-serious or reversible adverse effects associated with the use of TCM for CRF had been reported. Among the included studies, TCM which demonstrated significant benefits on the management of CRF could be either herbs  (n = 48), Ginseng Radix (人參) (n = 34) and Codonopsis Radix (黨參) (n = 30). From the perspective of TCM theory, these three herbs are the representative herbs of tonifying qi [Qi is the driving force of biological activity in the human body and plays a key role in body metabolism and immunity, which may help explain the mechanism of TCM in treating fatigue [qi deficiency and accumulation of dampness. Therefore, the herbs which promote and nourish the balance between yin and qi are often recommended in the treatment for CRF [Among the included studies in this review, more than 100 herbs, whether used as single herb alone or as a combination of herbs, were under investigation for their potential role in treating CRF. The three most common herbs which demonstrated benefits on CRF were fying qi . Qi is t fatigue . For can for CRF .Astragaloside IV, as an active extract of Astragali Radix, could inhibit the growth and proliferation of tumor cells and induce apoptosis of tumor cells [Ginsenosides, the main active agents in Panax ginseng, could help boost immunity by altering the levels of various cytokines [Ginseng Radix was associated with reduced inflammatory processes and released of cortisol through the stress axis and therefore could benefit CRF [Codonopsis Radix had the characteristics of Gan and Ping, tonifying the spleen and lung, nourishing blood, stimulating fluid, and strengthening body’s resistance [Codonopsis Radix, and few significant toxicity and side effects had been observed or recorded [In terms of the pharmacological mechanism, the two major pathological reactions of CRF are inflammatory immune and metabolic disorders . Similaror cells , and proor cells . Ginsenoytokines , 118. Itefit CRF . Codonopsistance . As reposistance . Toxicolrecorded .Kangai Injection (康艾注射液), followed by Buzhong Yiqi Decoction (補中益氣湯) and Shenqi Fuzheng Injection (參芪扶正注射液). These products composed of the three herbs mentioned above , and their benefits could be partly explained with the functions of tonifying qi and supporting righteousness, enhancing the body's immunity and improving fatigue.In addition to some herbs, a variety of TCM products were investigated for their role in the treatment of CRF. Among the TCM products which showed efficacy, the most researched products were Kangai Injection, a famous TCM injection, mainly consists of Astragali Radix, Ginseng Radix and Sophorae Flavescentis Radix. Network pharmacological indicated that some of the active extracts, such as oxymatrine, ginsenoside, and kaempferol, could regulate the pharmacological activity of TP53, TNF, VEGFA, EGFR or other key targets, and also mediate cancer, TNF, HIF-1, PI3K-Akt and some signaling pathways [pathways , 123. Itpathways . The propathways , 126 witpathways .Buzhong Yiqi Decoction  mainly composes of eight herbs including Astragali Radix, Atractylodis Macrocephalae Rhizoma, Ginseng Radix, Glycyrrhizae Radix, Angelicae Sinensis Radix, Bupleuri Radi, Citri Reticulatae Pericarpium and Cimicifugae Rhizoma. As a traditional Chinese decoction, it mainly played the role of regulating the spleen and stomach and ascending qi and yang [Buzhong Yiqi Decoction had strong immunomodulatory and anticancer effects [and yang , 129. In effects , 131. Ba effects .Shenqi Fuzheng Injection mainly composes of Astragali Radix and Codonopsis Radix [qi and support righteousness mostly for symptoms caused by deficiency of the lung and spleen. It can also be considered as an adjuvant therapy of lung cancer, breast cancer and colorectal cancer [is Radix  and has l cancer –136. Impl cancer , 137.Kangai Injection, Buzhong Yiqi Decoction and Shenqi Fuzheng Injection are the TCM prescriptions worth considered for their role and potential benefits in treating CRF.Similarly, this systematic review showed that these three TCM products might have a positive effect on CRF by improving some fatigue scale scores, cytokines level and blood tests compared with control group, and most patients showed no obvious adverse reaction after medication. Some findings also supported the anti-fatigue efficacy of these products in CRF patients , 138. AcAmerican ginseng (Panax quinquefolius) and Guarana (Paullinia cupana). Third-degree adverse reactions with American ginseng have been reported in rates ranging from 1 to 5% for agitation, insomnia, nausea, and vomiting [Guarana, depending on the different intervention doses and outcome measures, this herb might be effective or ineffective in alleviating fatigue symptoms [qi could damage the vitality of the body, produce dryness and heat, and cause symptoms of “Shang huo” in TCM theory [Apart from efficacy, the evaluation of the safety of TCM was another aspect of this review. Although the current evidence that some herbs improve the fatigue in cancer patients is strong, some herbs, even when used alone, should be considered with caution, such as vomiting . For Guasymptoms . Currentsymptoms . For exaM theory . The risM theory –144.However, compared to other TCM preparations, the risk of adverse effects associated with the use of injections was more concerning . AdverseDue to the insufficient evidence and high or unclear risk of bias of included RCTs, recommending TCM for CRF patients is a topic worthy of consideration. Further analysis of trials included in this review identified common limitations and provided relevant insights for designing and improving the quality of RCTs.Firstly, the overall quality of RCTs studies included in this review was generally poor, a finding commonly reported in previous systematic reviews involving TCM for other conditions –150. AmoSecondly, the limited sample size of included RCTs is also a concern. In this review, only 3 trials (3.66%) had more than 200 participants in the clinical studies , 33, 63 Thirdly, most of trials included in this review treated scale score changes as an outcome indicator to estimate the efficacy of using TCM. More than half of trials considered PFS (47/82) and QoL scales (51/82). Some other types of scales, including TCM syndrome score and fatigue scales, were also widely used. Currently, researchers aim to investigate more evidence for CRF, and a large number of fatigue scales are gradually appearing in the current research to assess psychological, physiological, endocrine, metabolic and other indicators of patients , 155. BuThere were several limitations in this review. Firstly, we only focused on RCTs, and excluded other study design and intervention approaches, resulting in limited consideration of the evidence for the treatment of CRF. Secondly, this review analyzed data from primary sources and the quality of trials was found to be low upon evaluation. There was some risk of bias in trials investigating the evidence on the efficacy and safety of TCM for CRF. Thirdly, no accurate diagnostic criteria for CRF patients were considered as multiple and incomplete diagnostic criteria were reported in the included RCTs. Finally, in TCM, there is often more than one ingredient to exert the therapeutic effect. The mutual induction or inhibition of various ingredients will also have an impact on the final curative effect. In our study, however, we only examined a few representative herbs and lacked an in-depth exploration of the inner relationship among other ingredients, which could result in bias when suggesting the herbs and products. Therefore, further studies need to improve on the quality of evidence, to establish the network analysis between relevant herb-ingredient-pharmacological activity of TCM for CRF, and to emphasize more on the efficacy of TCM.Based on further analysis of RCTs included in this review, common limitations and improvement were identified. The following summarizes insights into developments in the field of TCM treatment of CRF. (1) In order to standardize the use of TCM in the diagnosis and treatment of CRF, unified comprehensive diagnosis and efficacy measurement standards should be established; (2) multicenter, large-sample RCTs of CRF should be performed to minimize bias caused by age and sex differences and to ensure the validity of the results; and (3) clinical studies of multicomponent therapies should be conducted to enrich the TCM studies of CRF and at the same time verify whether it has a comparative advantage over a single components in terms of safety and reliability.Based on this review, the included TCM approaches have shown to be beneficial in the management and treatment of CRF. However, no recommended conclusion on the efficacy and safety of TCM in the treatment of CRF patients could be drawn as there were some concerns over the quality and bias of relevant RCTs. It is fundamental to standardize the diagnosis and treatment of CRF, improve RCTs protocol and conduct more clinical studies to present convincing evidence and provide an updated reference for disease medication in the future.Additional file 1. A detailed description of each search strategy.Additional file 2: Table S1. Level of risks of bias. Table S2. Diagnosis and included criteria of the CRF participants in RCTs in this review. Table S3. Evaluation of included trial studies using the CONSORT-CHM statement. Figure S1a. Risk of bias summary: review authors' judgements about each risk of bias item for each included study (Study No. 01-42). Figure S1b. Risk of bias summary: review authors' judgements about each risk of bias item for each included study (Study No. 43-81).


Clostridium autoethanogenum protein (CAP) is an economical and alternative protein source. Here, three experimental diets were formulated with CAP replacing 0% (CAP-0), 30% (CAP-30), and 60% (CAP-60) of fishmeal to investigate the alterations of structure integrity, fatty acids profiles, and lipid metabolism in the muscle of pearl gentian grouper. With increasing levels of CAP substitution, the percentages of 16 : 0 or 18 : 0 were decreased in triglycerides (TG) and diacylglycerols (DG); 18 : 1 or 18 : 2 was increased at the sn−1 and sn−2 positions in phosphatidylethanolamines; 20 : 5n−3 was increased in TG and DG. The phosphatidylcholines (PC) (18 : 3/20 : 5), PC(22 : 6/17 : 1), and sphingomyelins (d19 : 0/24 : 4) were identified as potential lipid biomarkers between CAP treatments. The CAP-30 treatment enhanced lipolysis and lipogenesis, while the CAP-60 treatment inhibited lipogenesis. In conclusion, fishmeal replacement with CAP affected the lipid characteristics and lipid metabolism, whereas it did not affect the structural integrity and fatty acids profiles in the muscle of pearl gentian grouper. Epinephelus lanceolatus♂×Epinephelus fuscoguttatus♀) is a hybrid seawater fish created by the Golden Seed Project to cultivate high-quality species with fast growth, strong disease resistance, and delicious meat [The pearl gentian grouper  [Micropterus salmoides) [Litopenaeus vannamei) [Acanthopagrus schlegelii) [in (CAP) , 10. Recin (CAP) . Studieslmoides) , 11, 12;annamei) –15; altelegelii) . HoweverLipidomics is a scientific method based on mass spectrometry, which can identify and quantify the types, composition, and structure of thousands of cellular lipid molecules in organisms so as to accurately discover and clarify the changes in lipid types and lipid metabolism , 18. At α  was purchased from Proteintech Co. . The antibodies against SREBP1  and P-PPARα , please refer to our previous experiment [The basic diet contained 50% fishmeal, and add CAP to the basic diet instead of 0%, 30%, and 60% of fishmeal to form CAP-0, CAP-30, and CAP-60 diets, respectively . At the same time, methionine and arginine were added to the feeds of CAP-30 and CAP-60 to reach the same level as that of CAP-0 so as to meet the needs of pearl gentian grouper [periment .E. fuscoguttatus♀×E. lanceolatus♂, n = 300, bodyweight of 18.01 ± 0.82 g) were distributed into 12 plastic barrels (300 L) reinforced with glass fiber, with 25 fish per barrel, ensuring four replicates in CAP-0, CAP-30, and CAP-60 treatments, respectively. The acclimation, feeding, and rearing conditions of fish are described in our previous study [Juvenile pearl gentian grouper  analysis was used to compare and evaluate the possible microstructure changes. SEM analysis refers to the previous experiments , 23, incSupplementary 2 and 3. The precision and stability of each fatty acid are shown in Supplementary 4. The sample was ground and the supernatant was collected; then, hexane was added after esterification, anhydrous sodium sulfate was added to the supernatant, and the supernatant was centrifuged again and hexane was added. After methyl salicylate was added, part of the supernatant was determined by gas chromatography–mass spectrometry. Processed samples were determined on a Trance 1310  equipped with a Thermo TG-FAME capillary column . The mass spectrometer ISQ 7000  was used to perform this operation in full scanning mode (mass range m/z 40–500). The concentrations of each fatty acid were calculated according to the peak area of fatty acid to the peak area of internal standard substance.The quantification of fatty acids in muscle was determined in accordance with the previous methods . The staμm). After analysis by the parameters of the mass spectrometry , the lipid molecules were identified and quantified using Thermo Scientific™ and LipidSearch™ 4.1 SP2 software. The raw lipidomics data (accession number: MTBLS4807) were deposited in the MetaboLights database [Quantification of lipid molecules in muscle was performed according to the previous methods , 25. Adddatabase .Supplementary 5: fatty acid synthase (fas), srebp1, pparγ, adipose triglyceride lipase (atgl), pparα, acyl-CoA oxidase 1 (aco), delta-6 fatty acyl desaturase (fad6), elongase of very long-chain fatty acid 4 (elovl4), elovl8, fatty acid binding protein (fabp), uncoupling protein 2 (ucp2), and liver X receptor alpha (lxr). β-actin and 18s rRNA were used as reference genes to normalize the genes expression. The results of gene expression were calculated using the 2ΔΔCT− method [The details of total RNA extraction using 1 ml of Trizol , cDNA preparation using an Evo M-MLV reagent Kit with gDNA Eraser (Accurate Biotechnology (Hunan) Co., Ltd), and qPCR assays using SYBR® Green Pro Taq HS (Accurate Biotechnology (Hunan) Co., Ltd) are provided in our previous study , 27. PriT method , 29.α , P-PPARα (1 : 800), and GAPDH .The total protein extraction, quantification, SDS-PAGE gel electrophoresis, transferring, blocking, incubation, and visualization assay were based on our published methods , 30. WesShapiro–Wilk test and Levene test were used for the normality and homogeneity test of results, respectively . One-wayThe SEM sections of muscle tissue of the pearl gentian grouper were obtained and compared. As shown in Supplementary 6, the pooled quality control samples were closely clustered and separated from the treatment groups , which demonstrating the high stability of the system and reliability of the data. The OPLS-DA scores in positive and negative ion modes were R2X = 0.767, R2Y = 0.993, and Q2 = 0.874, while the validation plots provided intercept parameters of R2  and Q2  (Supplementary 6). The results of OPLS-DA showed that the lipid composition and structure of the three treatment groups were obviously separated. At the same time, CAP-30 and CAP-60 groups showed small intragroup variations, while CAP-0 group showed large intragroup variations. Based on these findings, we concluded that the addition of CAP resulted in a dramatic change in lipid profiles in the muscle of pearl gentian grouper.In this experiment, the method based on untargeted lipidomics was used to identify and identify the characteristics of total lipid composition and distribution in the muscles of pearl gentian grouper with different levels of CAP. As shown in Supplementary 7 and Oreochromis niloticus) that lipid class mainly included TG, DG, PC, PE, phosphatidylserine (PS), and phosphatidylinositol (PI) [Penaeus kerathurus), mantis shrimp (Squilla mantis), and Pacific white shrimp [Portunus trituberculatus) [A total of 1,108 lipid molecules of 30 classes were identified in the muscle of pearl gentian grouper across different groups. It was mainly composed of phosphatidylcholines , triglycerides , phosphatidylethanolamines , diacylglycerols DG, 9.18%), and sphingomyelins  (tol (PI) . PC and tol (PI) . The PC e shrimp , 33, whi%, and spculatus) , 34. In Supplementary 6). Consistent with the present study, the level of TG was not affected in CAP treatments (0%–58.2%) in serum of black sea bream [Cyprinus carpio var. Jian), respectively [Compared to CAP-0 group, the CAP-30 treatment significantly increased the levels of PI, PS, and sulfatide (ST); the CAP-60 treatment significantly decreased the levels of phytoceramides (PhytoCer) and monoglyceride (MG) while significantly increased the levels of PS, lysophosphatidylserine (LPS), and ST . There were 61 DLM among CAP-0, CAP-30, and CAP-60 groups, comprising by 11 PC, 11 SM, 9 PE, 7 PI, 7 DG, 7 alkylphosphatidylcholine (PC(O)), 3 TG, 2 alkenylphosphatidylcholine PC(P), 1 Cer, 1 PE(O), 1 PE(P): alkenylphosphatidylethanolamine, and 1 Sph (Supplementary 8). Curiously, these DLM were largely inconsistent with the differential lipids classes that we found earlier (such as PS and ST). The heatmap was used to provide an intuitive visualization of the relative levels of lipids molecules, which showing their abundance in comparison groups. As shown in In the present study, the lipid species with Biomarker is a defined characteristic that is measured as an indicator of normal biological processes, pathogenic processes, or responses to an exposure or intervention , 37; we n−9, 18 : 2n−6, 20 : 5n−3, 22 : 5n−3, and 22 : 6n−3), the percentages of fatty acids in PC, PE, TG, and DG (the four most abundant classes) were calculated and compared.The positional distribution of fatty acids in the side chains of lipid molecules greatly aects its nutritional values and role in energy metabolism . To furtsn−1, sn−2, and sn−3, respectively [sn−2 positions, while the monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) predominantly occupied the sn−1 or sn−3 positions in muscle, head, and viscera of tilapia [sn−1 and sn−3 position, whereas the MUFA tended to be located at sn−1 and sn−2 positions in the present study  increased the catabolism of SFA and decreased their deposition in pearl gentian grouper  [n−3 was only located at the sn−2 and sn−3 position in pearl gentian grouper  is ectively . For the tilapia . In contnt study . The pretabolism . Compare grouper . In addiammation , 39. In  tilapia , we founo salar) . In part grouper . In the sn−1 position [sn−1 or sn−3 positions than at the sn−2 position in pearl gentian grouper, which is similar to the finding in Nile tilapia [n−3 and 22 : 6n−3 were inclined to deposit at the sn−2 position in PC and PE classes in swimming crab and Nile tilapia [n−3 is most preferentially binds to the PE skeleton, followed by the PC skeleton [n−3 in PE has a more important physiological function [n−3 deposition, compared to TG, PC, and DG classes  the percentage of 20 : 5n−3 was increased in TG and DG; (4) 22 : 5n−3 was accumulated at sn−1 in PC and at sn−3 in TG, while reduced at sn−1 in TG.To sum up, replacing of fishmeal by CAP (0%–60%) influenced the positional distributions of fatty acids in muscle of pearl gentian grouper . With inμg/g (on average), dominated by 16 : 0 (482.67 μg/g) and 18 : 0 (310.51 μg/g)  and 22 : 6n−3 (184.81 μg/g). The concentrations of n−6 PUFA were 202.15 μg/g, predominating by 18 : 2n−6 (169.20 μg/g). MUFA, especially 18 : 1n−9, is a “healthy” fatty acid for human body, which can remove bad cholesterol and protect cardiovascular and cerebrovascular health, etc. [μg/g, dominated by 18 : 1n−9c (146.34 μg/g). Compared to the CAP-0 group, the concentrations of C18 : 1n−9T was significantly decreased in the CAP-30 and CAP-60 groups; the concentration of C22 : 1n−9T was significantly decreased in the CAP-60 group. These findings suggested that the muscle of pearl gentian grouper maybe a good source of 18 : 1 n−9 for human consumption, while dietary CAP inclusions weaken this benefit.In general terms, SFA are used as an available energy sources, while PUFA are used in the synthesis of eicosanoids in kinds of fish tissues . In the 51 μg/g) . The conth, etc. . The conSupplementary 9 and 10). The PCA, PLS-DA and OPLS-DA score plots, as well as the hierarchical cluster, all showed no significant separation or clustering between the CAP-0, CAP-30 and CAP-60 groups (Supplementary 6), suggesting that fatty acids compositions were not affected by the CAP substitution. Similar to our study, the composition of all fatty acids did not differ from the control diet at low CAP levels (0%–30%), and the composition of SFA and MUFA did not differ at all dietary CAP levels (0%–100%) in Pacific white shrimp [n−3 PUFA was significantly increased, while the level of n−6 PUFA was significantly decreased at high CAP levels (more than 70%) [Basing on the AUC (1 or 0), none of fatty acid was identified as potential biomarkers between CAP-0 and CAP-30 groups or CAP-30 and CAP-60 groups (e shrimp . Howeverhan 70%) . Overallpparα in largemouth bass [α protein and gene and the ratio of p-PPARα to PPARα protein  could enhance the lipolysis, lipogenesis, and fatty acids desaturation, while a high level (60%) could inhibit lipogenesis and fatty acids elongation in the muscle of pearl gentian grouper.It was reported that the biosynthesis of LC-PUFA from 18 : 2 by fad6 . Meanwhin of DHA . In addiost fish . In the In summary, we found that: (1) fishmeal replacement with CAP  did not alter the structural integrity of muscle; (2) CAP inclusion resulted in large alterations in lipid composition and positional distributions of fatty acids; (3) the levels of PS and ST classes increased significantly with increasing levels of CAP substitution; (4) PC(18 : 3/20 : 5), PC(22 : 6/17 : 1), and SM(d19 : 0/24 : 4) were identified as the potential lipid biomarkers between the CAP treatments; (5) CAP replacement did not affect the fatty acids compositions; (6) 30% CAP inclusion enhanced lipolysis, lipogenesis, and fatty acids desaturation, while 60% CAP inclusion inhibited lipogenesis and fatty acids elongation. These findings provided scientific evidences and novel insights into the nutritional values of dietary CAP in aquatic animals.