Rectus abdominis biopsies were collected during cancer surgery from 35 patients diagnosed with cancer. Thin-layer and gas chromatography were used for quantification of phospholipid fatty acids. Cutpoints for survival were defined using optimal stratification. Median survival was between 450 and 500 days when patients had arachidonic acid (AA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in muscle phospholipid below the cut-point compared to 720–800 days for patients above. Cox regression analysis revealed that low amounts of AA, EPA and DHA are risk factors for death. The risk of death remained significant for AA , EPA  and DHA  when adjusted for sex. Lower amounts of essential fatty acids in skeletal muscle membrane is a predictor of survival in cancer patients. These results warrant investigation to restore bioactive fatty acids in people with cancer.Emerging studies are reporting associations between skeletal muscle abnormalities and survival in cancer patients. Cancer prognosis is associated with depletion of essential fatty acids in erythrocytes and plasma in humans. However the relationship between skeletal muscle membrane fatty acid composition and survival is unknown. This study investigates the relationship between fatty acid content of phospholipids in skeletal muscle and survival in cancer patients.  Experimental studies of cancer show that arachidonic acid , eicosapentaenoic acid  and docosahexaenoic acid  regulate proliferation, apoptosis, cytotoxicity, metastasis and immune cell functions in cancer cell lines3. EPA and DHA improve efficacy of anticancer treatments, chemotherapy tolerability, and inflammatory profile of the host while attenuating skeletal muscle mass depletion5, although the quality of studies is limited6. Collectively, the metabolic properties of essential fatty acids acting on tumor and host may influence survival of cancer patients.Essential fatty acids incorporated into membranes influence cancer progression by altering production of lipid mediators, influencing gene expression as well as activating signal transduction molecules influencing carcinogenesis12. Specifically, a decrease in AA, EPA and DHA has been reported in the blood phospholipids of cancer patients compared to healthy individuals, independent of changes in caloric and fat intake15. Higher content of palmitoleic (16:1) and oleic acid (18:1) concurrent with a lower content of AA and linoleic acid (18:2n-6) has been observed in erythrocytes of patients with lung adenocarcinoma and small cell lung cancer compared to healthy controls7. Shorter survival of lung, pancreatic and colorectal cancer patients exhibiting lower levels of essential fatty acids in blood plasma has been observed16. Murphy et al. reported 59% lower AA, 26% lower EPA, and 40% lower DHA in plasma phospholipids of cancer patients who survived fewer than the median survival of 238 days compared to patients who survived > 238 days9. Having a greater proportion of EPA and DHA in plasma phospholipids associates with better prognosis  in pancreatic cancer patients compared to patients with lower proportions16. A recent study reported lower AA in serum phospholipids of colorectal patients who exhibited disease progression after one year follow up compared to individuals without tumor progression13. Overall, measures in plasma phospholipids suggest that depletion of essential fatty acids is associated with shorter survival in cancer patients.Alterations in phospholipid fatty acid composition of adipose tissue, erythrocytes, neutrophils and blood plasma occur in patients with lung, colorectal and pancreatic cancers20. EPA and DHA are lower in plasma phospholipids of patients with sarcopenia compared to patients with normal muscle mass9. The highest concentrations of EPA and DHA in plasma phospholipids were observed in lung cancer patients who maintained or gained skeletal muscle during cancer treatment9. Several studies have shown an association between attenuation of muscle loss and n-3 fatty acid supplementation, however, these studies assessed fatty acid composition of plasma only and not of the muscle membrane19.EPA and DHA supplementation attenuate depletion of skeletal muscle mass in individuals with cancer, a well-established prognostic factorNo investigation into the alteration in fatty acid composition of phospholipids of skeletal muscle in cancer patients has been conducted. Based on observations in blood phospholipids, reduced levels of AA, EPA and DHA in skeletal muscle phospholipids are likely to be associated with shorter survival of cancer patients. The objective of the study was to investigate the relationship between fatty acid content of phospholipids in skeletal muscle and survival in patients with gastrointestinal cancers who provided biopsy during surgery. It was hypothesized that patients with higher levels of essential fatty acids in muscle phospholipids would have a better prognosis.The Molecular Profile of Cancer Cachexia.The study was approved by the Health Research Ethics Board of Alberta-Cancer and abides by the Declaration of Helsinki principles. Patients undergoing elective abdominal surgery were consecutively approached between Jun 2015 and Sept 2017 to participate in tumor and tissue banking at a hepatopancreatobiliary surgical service in Alberta, Canada. Ninety three percent of patients approached agreed to participate. Patients provided written informed consent for muscle biopsy and tissue banking and patient information  from medical records. The release of 35 samples from the tissue bank for analysis, as well as patient information  from medical records, was performed under the auspices of Protocol ETH-21709: Rectus abdominis biopsies of 35 patients undergoing surgery for colorectal, pancreatic and other gastrointestinal tumors were studied. The study cohort and conditions for acquisition of muscle samples have been described previously21. Patient characteristics are shown in Table 22. CT scans completed before surgery were analysed using SliceOmatic V4.2 software with CT image parameters that include: contrast, 5 mm slice thickness, 120 kVP, and 290 mA. Total skeletal muscle area (cm2) was evaluated on a single image at the third lumbar vertebrae (L3) using Hounsfield unit (HU) thresholds of − 29 to 150 for skeletal muscle. Total skeletal muscle area was normalized for stature (m2) and reported as skeletal muscle index (SMI) (cm2 m2). Mean muscle radiodensity (HU) is reported for the entire muscle area .CT scans completed with a spiral CT scanner for initial cancer staging and routine diagnostic purposes were used to quantify muscle area, cross section area, adipose tissue area and mean skeletal muscle radiodensity to confirm that the population sampled is representative of mean values for cancer population2; 0.025%] solution. A modified Folch method was used to extract lipids from muscle12. Lipids were extracted using chloroform/methanol . Thin layer chromatography chromatography (TLC) plates  was used to isolate phospholipids (PL). Solvent  was added to the developmental chamber. Chloroform/methanol (2:1) was added to each dried tube, vortexed and samples were spotted on plates in duplicate. Spotted plates were run in a solvent system until the solvent mixture reached ~ 1.5 cm from the top of the plate. Dried plates were sprayed with 0.1% 8-anilino-1-naphthalenesulfonic acid (ANSA) to visualize the PL bands under ultraviolet light. Bands were scraped andan internal standard, C17:0, was added for PL fatty acid quantification followed by methylation. PL fatty acid composition was analysed by gas chromatography-flame ionisation detector in a Varian 3900 gas chromatography . The quantity of fatty acids within the PL fraction was calculated by comparison with the known concentration of the internal standard and the sum of all fatty acids was reported as total PL. The peak area of each fatty acid was normalized against the sum of peak areas of all fatty acids to determine the relative proportions of fatty acids comprising the PL fraction.The biopsy [≈ 50 mg] was ground using a frozen pestle and mortar without letting the muscle tissue thaw. Ground tissue was homogenized in calcium chloride [CaCl23. This method identifies cut-points, to establish a level of fatty acids that best separate patients at risk with respect to survival. It is appropriate to determine survival-related threshold values empirically using statistical methods such as optimal stratification, where the relationship with survival for that covariate is not known . In our study, we determine the cohort specific threshold amounts of phospholipid fatty acids; thresholds for these fatty acids were examined by BMI, age, tumor site and metastasis and sex. Number of days of survival were defined as days to death from the date of surgery or as number of days between date of surgery and date of data collections . The Kaplan–Meier method24 was used to establish the effect of the association between the quantity of phospholipid fatty acids on the number of days of survival. Log-rank tests were used to compare the survival curves for each variable (p < 0.05). Variables were entered into a univariate Cox proportional hazards model with 95% confidence intervals. Models were adjusted for BMI, age, tumor site and metastasis and sex. Statistical significance was reported when p-value < 0.05. Data are reported as mean ± SD. Levels of significance are p values < 0.05. All statistical analyses were performed using SPSS 20.0  for Windows.The primary outcome was overall survival, defined as the number of days surviving after the initial visit by each patient. Patients were observed until their deaths or until November 30, 2018, at which time they were censored at the last date they were documented to have been alive. For survival analysis, univariate Cox proportional hazard model was used to determine if amounts of fatty acids (continuous variable) were associated with the number of days of survival. Given the small sample size, fatty acids that were associated with the number of days of survival with p-value less than 0.1 were selected for optimal stratification and Cox regression analysis . Optimal stratification, a statistical method similar to receiver operator curve analysis, was used to solve specific threshold values within continuous variables. Optimal stratification is based on log-rank statistics that best separate patients with respect to time to an event outcome (death)The association of fatty acids with survival was first determined Table . UnivariThe survival distributions for patients below and above the cut-points for the essential fatty acids and adjusted for sex were statistically significantly different .9. Also, longer survival of pancreatic cancer patients has been associated with higher content of EPA and DHA, but not AA, in plasma16. Lower proportions of AA, EPA and DHA in plasma and erythrocyte phospholipids have been reported in bladder, lung, colorectal and pancreatic cancer as compared to healthy or non-malignant populations16. Proportions of AA, EPA and DHA in skeletal muscle in our cohort of cancer patients were lower compared to reference range of healthy person26. Here, we demonstrate a reduction in EPA, DHA and AA in muscle and further associate this depletion with truncated survival.In a cohort of patients with depletion of AA, EPA and DHA below a critical level as determined by statistical cut-points, approximately 40% more patients died compared to those with fatty acids above 136.9 ng/mg, 7.2 ng/mg and 25.2 ng/mg, respectively in muscle phospholipids. Notably, only one patient died in the group of patients with AA, EPA and DHA above this critical level but 600 days post-surgery. In contrast, over half of patients died within one year of surgery in the group of patients with AA, EPA and DHA below the critical level. Two studies have previously reported an association between levels of AA, EPA and DHA of plasma phospholipid and survival. Lower content of AA, EPA and DHA were reported in plasma of cancer patients who lived < 238 days as compared to > 238 days, determined to be the median days of survival in that study population28 and plasma phospholipid fatty acid depletion9 have each been separately reported to be predictors of survival in cancer. The positive association between skeletal muscle mass and concentration of EPA and DHA in plasma phospholipids has been previously reported9. Based on this, it was hypothesised that there would be a positive association between essential fatty acids and skeletal muscle index, however, this was not observed.Muscle loss31. The association between dietary/plasma AA and the risk of cancer is highly controversial32. AA and its metabolites play role in muscle growth33. AA is converted into two series eicosanoids known as prostaglandins F2alpha (PGF2α) and prostaglandin E2 (PGE2) which activate the major anabolic pathway in muscle, phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signalling pathway and induce myotube hypertrophy35. Suppression of PGF2α in the muscle of experimental models has been shown to inhibit muscle recovery after disuse atrophy36. EPA and DHA in skeletal muscle membranes have been suggested to influence membrane properties that may influence anabolic signalling, particularly through key regulators of muscle protein synthesis37, and given the association with survival, these mechanisms are worthy of exploration.A strong association is observed between essential fatty acids in phospholipid of muscle. Membrane phospholipid fatty acids regulate the biophysical properties of proteins, provide substrates for second messengers and intracellular signals to alter gene expression. Evidence suggests that EPA and DHA in membrane phospholipids can have a protective effect against cancer through several actions including protein kinase activation, enhancing cell apoptosis and modulating inflammation39 and food choices cancer patients make during their cancer trajectory are foods that are good sources of AA, and its precursor linoleic acid41, therefore, depletion of AA observed is not likely to be related to intake, but rather a change in metabolism of essential fatty acids. Moreover, studies reported alterations in plasma and erythrocyte fatty acid composition in cancer patients compared to healthy controls independent of total calorie and fat intake42. Second, reliability of optimal stratification in small sample size is low43. The thresholds determined in this study should be considered approximate until confirmed in additional studies with larger sample size. While the first and only study to evaluate the PL composition in relation to survival, the sample size is too small to adjust for several well-known prognostic factors, which should be accounted for in future studies. There are several covariates that can be considered in the oncology setting, including tumor stage which is known to impact survival44. We did not see significant association between tumor site/metastasis and survival in our cohort. This can be due to small sample size and there is a chance of type II error. Ours is the preliminary study that provides new information that can be used to adequately power a future study to adjust for all covariates.Several limitations of the current study warrant mention. First, dietary records were not available for the patients, therefore, the effect of diet on skeletal muscle composition was not determined. The western dietIn conclusion, patients with AA, EPA and DHA amounts in muscle phospholipids that were below a critical level had shorter survival. The present study is a first step in establishing alterations in skeletal muscle fatty acid composition and its association with survival in cancer. This work provides rationale for conducting further studies to examine repletion of bioactive fatty acids in cancer. Further work is needed to investigate mechanisms that can explain the strong associations between essential fatty acids and survival in cancer patients observed in this study.Supplementary Information.Supplementary Table 1.


In light of our controls, our data suggests that when molecular tools were used to characterize our cultivation efforts, they provided a more complete and more complex, understanding of which organisms were present compared to what was eventually detected during cultivation. Lastly, about 3% of the isolates collected from our cultivation experiments showed inhibitory bioactivity against an already multidrug-resistant pathogen panel, further highlighting the importance of informing and directing future cultivation efforts with molecular tools.Molecular techniques continue to reveal a growing disparity between the immense diversity of microbial life and the small proportion that is in pure culture. The disparity, originally dubbed “the great plate count anomaly” by Staley and Konopka, has become even more vexing given our increased understanding of the importance of microbiomes to a host and the role of microorganisms in the vital biogeochemical functions of our biosphere. Searching for novel antimicrobial drug targets often focuses on screening a broad diversity of microorganisms. If diverse microorganisms are to be screened, they need to be cultivated. Recent innovative research has used molecular techniques to assess the efficacy of cultivation efforts, providing invaluable feedback to cultivation strategies for isolating targeted and/or novel microorganisms. Here, we aimed to determine the efficiency of cultivating representative microorganisms from a non-human, mammalian microbiome, identify those microorganisms, and determine the bioactivity of isolates. Sequence-based data indicated that around 57% of the ASVs detected in the original inoculum were cultivated in our experiments, but nearly 53% of the total ASVs that were present in our cultivation experiments were  Molecular techniques and sequencing technologies have helped to describe many microbial communities and provide tools that allow us to study representative microorganisms and their activities in lieu of isolation. Application of molecular approaches to the study of microbiology have led to the discovery and characterization of rare biospheres of the most extreme environments  and enabIsolating, or even just cultivating representative microorganisms is still considered the best means toward understanding their physiology, metabolism, and potential ecological roles, but this remains to be a major hurdle. The degree to which any microbial community is represented in culture varies considerably, though this has been the topic of debate . Since SEfforts to isolate yet uncultured microorganisms have gathered momentum as researchers have begun to apply new technologies and innovations . MicrobiWhen cultivation approaches are coupled with molecular analyses, the total diversity and identity of cultivated taxa can be determined , and speStreptomycetes spp. . The goals for this study were to (1) compare the diversity of cultured bacterial communities on multiple types of media combined with various treatments of the inoculum, (2) determine which media, treatments, or combination cultivated the highest richness of bacterial taxa, and (3) determine how many isolates produced bioactive molecules against a panel of multi-drug resistant pathogenic microorganisms.Procyon lotor) deceased less than 8 h was sampled in triplicate (n = 3) with sterile, nylon swabs. One concern was that swabbing for sequencing first would remove or alter the biomass when the cultivation swab samples were taken. To avoid this, each orifice was swabbed three times. At each of those times, two swabs were used, one destined for cultivation the other destined for sequencing. In doing so, any alteration of the biomass would be reflected in each set of triplicate swabs, for sequencing and for cultivation. This generated a total of 6 swabs from each orifice, 3 for sequencing and 3 for cultivation. Swabs to be used for cultivation were pooled by orifice and stored in a sterile conical tube containing 3.0 mL 1X PBS, stored on ice, and immediately transferred back to the laboratory (under 45 min). Swabs to be used for sequencing were transferred to separate, empty, sterile conical tubes to preserve the technical replicate, stored on ice, and immediately transferred back to the laboratory (under 45 min). At the laboratory, swabs for sequencing were transferred to BashingBead Lysis Tubes  containing 750 mL of BashingBead Buffer (Zymo Research Corp.). Cells were lysed through homogenization in a Mini-BeadBeater-8  at maximum speed for 45 s. Homogenized samples were then stored at −20°C until needed for DNA extraction. Swabs for cultivation were vortexed to suspend cells. A 900 μL aliquot of the cell suspension intended for storage was centrifuged at 10,000 × g for 1 min, the supernatant was removed. The cell pellet was resuspended in 750 mL of BashingBead Buffer (Zymo Research Corp.) and transferred to BashingBead Lysis Tubes  prior to lysis through homogenization in a Mini-BeadBeater-8  at maximum speed for 45 s. Archives were stored at −80°C. The remaining 2.1 mL of the cell suspension for each sample was serially diluted with PBS. Aliquots of 50 μL from the 100–10–6 dilutions were spread onto 13 different media/treatment combinations . Each sample was transferred to a BashingBead Lysis Tube (Zymo Research Corp.) and homogenized for 45 s at maximum speed using a Mini-BeadBeater-8 . The lysed samples were stored at −20°C until DNA was extracted.Quick-DNA Fungal/Bacterial Miniprep kit . For community characterization, a conserved region (V4-V5) of the SSU rRNA gene of most bacteria, archaea, and eukarya was amplified using primers 515F-Y and 926R (GTA AAA CGA CGG CCA G CCG TGY CAG CMG CCG CGG TAA-3′) contains the M13 tag (underlined) fused to the 5′ end of the forward primer . DNA was extracted according to manufacturer specifications using the Zymo and 926R  via the d primer . The revdada2 pipeline . Concerning ASV filtration, the dada2 pipeline does not infer an amplicon sequence variant that is only supported by one read, therefore, the final ASV table does not contain singletons, which could affect statistical methods for richness (though no statistics were performed on richness in this study). Lastly, chimeras were removed  and taxonomy was assigned using the SILVA database v32 (Barcodes from amplified regions of the 16S rRNA gene were removed and demultiplexed using QIIME v 1.9.1 . Demultipipeline  and amplbase v32 . SequencPRJNA675861.Sequence data has been deposited at NCBI’s Sequence Read Archive (SRA) database under accession number phyloseq . Differences in cultivable diversity could be due to different starting communities from each orifice. So, orifice was treated as a fixed factor to control for any influence it could have on the significant differences in diversity between cultivable communities. To fully understand the significance described in the permanova, a permdisp with the betadisper function in vegan was used to describe any within sample variance  that could explain any significant differences detected in the permanova. Hypothesis testing via ANOVA with permutations (n = 999) was used with permdisp to determine any significant differences in variation within samples. These statistical methods allowed us to determine if our cultivation strategies cultivated diverse subsets of organisms that were different from each other between media and treatment types.Alpha diversity was calculated as richness of ASVs, while beta diversity was measured with non-metric multi-dimensional scaling (NMDS) using Jaccard distances. A permanova with the orifices . Due to indicspecies package (permutations = 999)  . Species10 CFUs/mL cultivated across all medium types and dilutions, a total 238 colonies were chosen for isolation based on differential colony morphology from the mouth and rectum on a subset of the media types . Colonies were isolated by streaking for isolation of single colony morphologies at least three times before the assumption of pure cultures. To determine bioactivity, soft agar overlays in 0.1X TSA (0.8% agar) were used to test for growth inhibition of a panel of drug-resistant pathogens . To prepare for the bioassay, pathogens were incubated overnight, shaking at 250 rpm at 30°C, in 0.1X tryptic soy broth (TSB) . A 400 μL aliquot of each pathogen was used to inoculate 3.6 mL of molten TSA soft agar (at 55°C), mixed, and immediately poured onto a 0.1X TSA agar plate for a final overlay volume of 4 mL. Colony material from each isolate was transferred to the bioassay plate with a sterile toothpick by etching an “X” into the overlay. The inoculated bioassay plates were incubated at 30°C for 24 h. Inhibition of the pathogen in the overlay was characterized by a zone of clearing surrounding the isolate  according to manufacturer’s protocol from the above mentioned 0.1X TSB cultures. The SSU rRNA gene was amplified using primers 8F (3′-ATGC-5′) and 1492R (3′-ATGC-5′) at a concentration of 0.2 mM, 1 × 5 PRIME HOT master mix , and 2.0 mL of DNA template per 50 mL reaction via the following PCR protocol: initial denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 45 s, and extension at 72°C for 45 s, with a final extension at 72°C for 10 min. Amplified fragments were purified using Sera-Mag magnetic beads (GE) with the AmPureXP (Beckman Coulter) protocol at a final concentration of 1.8x v/v. Some isolates were not recoverable after the process of freeze-thawing, and some extractions did not result in enough DNA for sequencing. Purified amplicons  were sent for Sanger sequencing using the 8F primer . Resulting sequences and chromatograms were assessed for quality using Integrative Genomics Viewer (IGV) . SequencAs expected, a library of SSU rRNA genes from the original inocula showed that they had higher richness  than the cultivated organisms from both orifices . The micp < 0.05) associated with orifice, medium type, and/or treatment. Only one family, the Aeromonadaceae, was significantly associated with a particular orifice, the mouth. This family was also the only family significantly (p = 0.001) associated with a certain base medium type, ROXY (p < 0.05) associated with a certain treatment or combination of treatments . Based on molecular analyses, 27 families were shared between the mouth and rectum before cultivation, while 6 families were unique to the mouth and 6 families were unique to the rectum . NegativSerratia (34.2%) and Klebsiella (17.9%) . Despite choosing colonies based on differential morphology, many of these isolates were expected to be redundant, since the diversity of the initial inoculum was low . This wa (17.9%) . Each ofidentity .adonis2 function in the vegan package , while base medium type alone explained only ∼5% of the observed variation . The interactions of base medium and treatment were insignificant and did not fit the model . An analysis of variances (permdisp) was used to determine if any significance was driven by a dispersion effect rather than a location effect for orifice, base medium type, treatments, and medium + treatments. Mean distances to centroids did not differ significantly for orifice  or medium + treatment , meaning little inter-sample variation was detected between replicates. In contrast, the mean distances did differ for treatment  = 0.50, catalase = 0.53, ethanol = 0.49, streptomycin = 0.56) and base medium type . The significance in dispersion within the treatment and media could correspond to the significance in permanova results, where much of the variation explained in the permanova may be due to the intrinsic variability and stochasticity among replicates of the same inoculum.Due to the nestedness of this experiment , the stratification of taxa based on sampled orifice was corrected for hypothesis testing with permanova by defining orifice as a stratum in the  package . The majProcyon lotor) to (1) compare the diversity of cultured bacterial communities on multiple media types and/or treatments of the inoculum, (2) determine which media/treatments cultivated the highest richness of bacterial taxa, and (3) determine if any isolated organisms produced bioactive molecules against a drug resistant pathogen panel. The occurrence of antimicrobial resistance is increasing at a faster rate than new therapies are entering the market , or that were streptomycin resistant. These treatments had a greater effect on the cultivable diversity compared to changes in base medium alone, likely because the base media shared many of the same oxidizable substrates. It is logical to hypothesize that the inoculum exposed to selective treatments would have decreased cultivated richness compared to the same inoculum on an untreated medium of the same type, since the selective agent would inhibit the growth of a portion of the community. Instead, the cultivable richness and diversity varied for the same treatments on different base medium types and between orifices . This con = 3), screened for antimicrobial production and identified through SSU rRNA sequencing. Colonies were chosen based on differential morphologies. Overall, 7 of the 238 recovered isolates showed bioactivity against the already antibiotic-resistant pathogen panel, resulting in a ∼3% hit rate. Five bioactive isolates were sequenced. Three belonged to the phylum Proteobacteria (class Gammaproteobacteria) and two belonged to the phylum Firmicutes. Two isolates were identified as a Bacillus sp. and a Pseudomonas sp., both genera known to contain species that produce bioactive compounds  consistently growing on replicate plates .Neither differences in cultivation strategy nor orifice sampled could explain the majority of the observed variation in community structure . We can Cultivation on agar plates warrants special consideration when discussing diversity. The relative distribution of populations on an agar surface affects their prevalence and the overall composition of the cultivated community. Further, the physiology and colony morphology of each population dictates their prevalence and that of the surrounding populations on an agar surface. Colony size, especially surface area, is impacted by traits such as growth rate, surface motility , presence of inhibitory metabolites, and the availability of resources. These differences in biomass distribution were important when deciding how to measure diversity, a critical metric for assessing cultivation on an agar surface. Unlike liquid cultures, varying biomass  from different taxa that might produce an equal number of colonies can over-estimate the relative abundance of that taxon, studies relying on relative abundance to characterize diversity should consider this . If a poIn this data set, about half of the ASVs detected after cultivation were not detected in the molecular controls. This same phenomenon was observed by Zehavi et al. when cultivated rumen OTUs outnumbered the OTUs detected in the original rumen sample and its dilutions  . This diCultivation is critical to answer broad challenges of microbial ecology like deciphering microbial metabolisms or specific challenges like obtaining new isolates for antibiotic discovery. The study of pure cultures remains the best approach to comprehensively describe an organism’s physiological and metabolic properties, and efforts to optimize cultivation strategy have proven successful . Our datThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/EJ conceived and conducted the experiments, analyzed data, and wrote the manuscript. BS conceived the experiments, analyzed data, and wrote the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Theobroma cacao L.) is sensitive to water deficit stress (DS), which limits productivity. The aim of this research was to characterise the response of seven  commercially important cacao clones to severe and temporal water deficit stress. Ten-month-old cacao trees were submitted to two treatments: well-watered and water-stressed until the leaf water potential (Ψleaf) reached values between −3.0 and −3.5 MPa. The effects of hydric stress on water relations, gas exchange, photochemical activity, membrane integrity and oxidative stress-related gene expression were evaluated. All clones showed decreases in Ψleaf, but TSH565 had a higher capacity to maintain water homeostasis in leaves. An initial response phase consisted of stomatal closure, a general mechanism to limit water loss: as a consequence, the photosynthetic rate dropped by approximately 98% on average. In some clones, the photosynthetic rate reached negative values at the maximum stress level, evidencing photorespiration and was confirmed by increased intracellular CO2. A second and photosynthetically limited phase was characterized by a drop in PSII quantum efficiency, which affected all clones. On average, all clones were able to recover after 4 days of rewatering. Water deficit triggered oxidative stress at the early phase, as evidenced by the upregulation of oxidative stress markers and genes encoding ROS scavenging enzymes. The effects of water deficit stress on energy metabolism were deduced given the upregulation of fermentative enzyme-coding genes. Altogether, our results suggest that the EET8 clone was the highest performing under water deficit while the ICS-60 clone was more susceptible to water stress. Importantly, the activation of the antioxidant system and PSII repair mechanism seem to play key roles in the observed differences in tolerance to water deficit stress among clones.The increase in events associated with drought constraints plant growth and crop performance. Cacao ( Theobroma cacao L.) is a fruited tree native to the Amazon basin of South America  or maize (Zea mays L.)  . Howevermays L.) . Howevermays L.) . With thThis study was carried out at Bambusa Station (Geoambiente SAS), which is located in the dry premontane forest at 5°07′50″ north and 74°09′30″ west and 1,304 m.a.s.l. in Pacho (Cundinamarca-Colombia). All clones were propagated in a glasshouse by grafting and using Caucasia (Colombian) rootstock (Geoambiente SAS) for mass production. Ten-month-old well-developed seedlings were used for the experiment. The commercial clones EET8, FEAR5, CCN51, TSH565, ICS1, ICS60, and ICS95 were selected because they belong to the group of the most recommended materials for planting and marketing in Colombia by the National Cacao Council  and base® (Fundases) and 5 g of 15-15-15 (N-P-K) Nutrimon® (Monómeros Colombo Venezolanos S.A) per liter of water. This fertilization scheme was applied twice after grafting to ensure the mineral requirements of the plants for optimal growth and development. From the time of sowing to the application of water stress treatment, all plants were irrigated to field capacity. During the evaluation period, the maximum and minimum temperatures and relative humidity were registered daily with a HOBO 8 weather station (HBOware®) installed at 0.50 m above the ground (®). Under field capacity, the predawn foliar water potential (Ψleaf) remained between −0.2 and −0.4 MPa according to a previously reported Ψleaf value for well-irrigated cacao plants  was established as the maximum water deficit stress point  of silty loam soil (pH 6.5). Considering the results of the soil analysis, each plant was fertilized with 5 mL of Agropluse ground . The meae ground  was alsoe ground  were plae ground . Consequo plants . In the −3.5 MPa  moment i−3.5 MPa , and thess point . At the leaf value was measured pre-dawn from 5:00 to 6:30 am in 3 or 4 completely expanded leaves from the top to the bottom of six plants per treatment (n = 6) and for each clone  and intercellular CO2 (Ci) were registered from 9:00 am to 12 m in 3 or 4 completely expanded leaves from top to bottom of nine plants per treatment (n = 9) and per clone using a LI-6400XT Portable Photosynthesis System measurement system  with an ambient CO2 concentration (Ca) of 400 μmoles m–2 s–1 (–2 s–1 according to the response of photosynthesis to light for each clone (data not shown). The intrinsic water use efficiency (WUEi) was calculated with A and gs data (A/gs).The parameters net photosynthesis (A), stomatic conductance (g m–2 s–1  and a phv/Fm) was measured in dark-adapted leaves for 45 minutes and the effective quantum yield of PSII (Y[II]) was measured during a repetitive saturating actinic light pulse equivalent to the ambient light (n = 9). For this, the chlorophyll molecules were excited for 0.80 s with 1,500 μmoles m–2 s–1 actinic light. Measurements were realized from 4:00 to 7:00 pm, using a Junior-PAM modulated fluorometer  and the parameters were calculated with WinControl-3 software   .The RWC leaf was determined following the protocol proposed by 2 of 21 plants per treatment. The turgid weight was determined after hydrating the pieces with distilled water for 24 hours, and the dry weight was then recorded after drying the pieces in a 70°C oven to a constant weight.The leaf fresh weight, turgid weight and dry weight were determined in leaf pieces of 25 cm® - ICT, SL, Bogota, Colombia) after 6 h of incubation and after heating the same sample at 90°C for 2 h. EL was expressed as a percentage (%) according to the equation proposed initially by The electrolyte leakage (EL) was determined using the protocol proposed by max is the EC measured after heating the sample at 90°C for 2 h.where, EL is the percentage of lost electrolytes, EC is the EC measured at 6 h, and ECAmong the seven clones evaluated, three clones were selected as representatives (see below) of the different responses to DS observed through the physiological variables evaluated. Samples of mature foliar tissue from three plants of each clone were taken on day 0 (control), day 23 and day 26 after DS, once physiological measurements completed (noon) and immediately frozen in liquid nitrogen for total RNA extraction.A search for genes associated with the water stress response (drought or waterlogging) and oxidative stress (antioxidant system) was performed for model plants in the literature and in sequence databases. Then, the selected CDSs . Finally, a total of nine primer pairs were designed for the respective cacao orthologous genes and three pairs were designed for the housekeeping genes . High RNA yield (∼500 ng/μl) with an RNA integrity number (RIN) higher than 7 was obtained after optimization of the protocol. Reverse transcription was performed with 1 μg of total RNA using M-MuLV Reverse Transcriptase  following the manufacturer’s instructions and using oligo d(T) 18 primers.As mentioned above, all harvested leaf samples were maintained in liquid nitrogen and stored at −80°C until RNA extraction. For RNA extraction, an in-house protocol was optimized based on the protocol of ® . Amplification assays were performed in a Quant Studio 3 Real-Time PCR System . The specificity of all amplifications was verified using melting curves and the threshold cycle values (Ct) of the evaluated and housekeeping genes were registered to calculate the changes in relative expression using the 2–ΔΔCt method . All amplifications included negative template controls run in triplicate. RT-qPCR was performed using a volume of each cDNA equivalent to 5 ng of reverse-transcribed total RNA and 0.25 μM of each primer pair in 15 μl of final reaction volume using Luna Universal qPCR Master Mixt method . Previout method  were obtaluated. . Detailea posteriori tests (P < 0.05). The analyses were carried out using R software (R Studio Version 1.1.463)  were performed to determine the effect of the clones and treatments on the physiological variables registered and whether there were significant differences in the relative gene expression of selected genes between clones and days after suspension of irrigation. Subsequently, a mean comparison analysis was performed to assess statistically significant differences through LSD and Tukey’s HSD 1.1.463) .The air temperature in the glasshouse during the experiment ranged between 17 and 35°C, with a mean value of 26°C, and the average relative humidity was 93% . Furtherleaf value was significantly different between water states at D26 and between DS plants  while the clone with the lowest Ψleaf was ICS95 (−2.83 MPa). Interestingly, at 4 DAR, all clones previously submitted to DS showed similar Ψleaf values to their respective control (WW) clones (between −0.183 and −0.266 MPa)  . TherefoThe RWC maintained high values in all WW clones (between 86.36 and 91.33%). At D26, the FEAR5, ICS60, ICS95 and TSH565 clones experienced a significant decrease in RWC , with FEIn contrast, EL did not show significant differences between WW and DS in most of the clones, showing a value of 16% on average under DS, similar to all WW plants, except for the ICS1 and ICS60 clones, for which significant increases in EL upon DS were observed, reaching 23.1% and 23.4%, respectively .s, while Ci was increased significantly in all the clones evaluated  at D26, reaching negative values and ranging between −0.09 and 0.086 μmoles of CO2 m–2s–1, without significant differences between clones. The negative values are indicative of net CO2 production in the light , which is also supported by the higher Ci concentration found under DS. Interestingly, despite reaching A values close to 0 μmoles of CO2 m–2s–1 at DS, the ICS1 and EET8 clones did not show negative values, even at D26  corresponded to ICS95 and FEAR5, for which the increase was 69.1 and 54.2%, respectively, and the lowest values were found for the clones CCN51 and ICS1 (356.8 and 358 μmol CO2 mol–1), reflecting an increase of approximately 30%. Notably, significant differences were found between the clones presenting the lowest values and those showing the highest values of Ci  was observed upon DS treatment in most of the clones compared to their respective WW controls. WW gs values varied from 0.057 to 0.089 mol of H2O m–2s–1, while under DS, the gs values ranged from 0.024 to 0.008 mol of H2O m–2s1.Furthermore, a significant reduction in gi was also reduced meaningfully under DS in most of the clones and reached negative values, as observed for A. Under WW conditions, the WUEi varied between 60 and 69 μmoles of CO2 mol H2O–1, whereas under DS, the values ranged from 7.150 to −49.42 μmoles of CO2 mol H2O–1. Importantly, under DS, WUEi was significantly higher for clones ICS1 and EET8 than for the other cacao clones that showed negative WUEi , and the lowest Fv/Fm values  were significantly different from EET8 and CCN51  clones without significant differences between them at 4 DAR . In genev/Fm parameter or Y[II], while the ICS60 clone was one of the most affected clones in these two parameters together with a significant increase in EL, as stated above. The impact of these three parameters, particularly Fv/Fm, could reflect photoinhibition by oxidation, assessing the existence of this condition to be relevant in T. cacao. Therefore, to determine both the extent of this probable oxidative stress induced under DS and whether different cacao clones responded differently regarding the activation of their antioxidant system, gene expression profiling was carried out on three selected cacao clones: the two most contrasting clones with regard to their photosynthetic and chlorophyll fluorescence parameters, as well as the EL parameters (EET8 and ICS60), together with the TSH565 clone, which maintained the highest water potential under DS.Among the differences in the response to DS observed between clones, interestingly, the EET8 clone did not show a decrease in either the FT. cacao  croT. cacao , and (3)T. cacao . FurtherGST expression profiles, at D26, EET8 was the only clone to maintain induction of both GST and HSP genes compared to both ICS60 and TSH565 clones, which showed a downregulation pattern at the maximum level of DS (D26). In contrast, for the HSP gene, the upregulation observed at D23 was significantly higher in ICS60 and TSH565 clones than in EET8 clones. Although there were few differences in the expression profiles between clones, the strong induction of both oxidative stress markers observed at D23 before the maximum level of stress suggests that DS triggered oxidative stress in all cacao clonesSignificant differences in the expression patterns of these two oxidative stress markers were found after water deprivation in all three clones evaluated . InteresRBOHF, NTRC, UDPGT, and SOD) were analyzed according to their expression profile in response to DS in cacao. In general, all genes showed a similar pattern to that found for GST and HSP, i.e., a significant induction at D23 in all clones  . However, the TSH565 clone showed the highest Ψleaf value (−2.07 MPa), indicating that it was less affected than the rest of the clones  . In thise clones , which st stress . Moreoverigation . García −1.3 MPa  is a dir−1.3 MPa . Indeed,W plants .In addition, it has been observed that reduced water supply impacts cacao growth and development through a reduction in the leaf area, which leads to lower net assimilation, stomatal conductance, and yield, being this reduction of leaf area considered one of the earliest acclimation mechanisms to withstand drought . Impact s or A in the plants, since the normal stomatal closure values in cacao are close to 1.8 KPa as reported previously  were evaluated , and thece trait .As mentioned above, the observed stomatal closure allowed it to keep the RWC above 80% in all clones at D26 under DS, thus maintaining leaf turgor and preventing tissue damage, which is a known mechanism of DS avoidance. Similar responses have been found in other cacao clones exposed to water deficit without a reduction in RWC . This abi, a variable that relates A and gs  observed for the EET8 clone compared to ICS60.In addition to and related to the relationship between carbon fixation and stomatal closure, the Cv/Fm and Y[II] values upon DS in clones that exhibited a lesser tolerance based on water status or gas exchange parameters . In contrast, stressed EET8 clones showed Fv/Fm and Y[II] values similar to those obtained in their cognate WW controls. Fv/Fm reduction has been related to a photoinhibition phenomenon, with the concomitant reduction in Y[II] and increase in Ci. The cacao response to DS may therefore have two limiting phases depending on the genotype: one initial and stomatal-controlled phase, resulting in a substantial reduction in A and Ci as gs decreased, followed by a second and strongly photosynthetically limited phase that caused an increase of Ci while Fv/Fm and Y[II] decreased as an indication of the onset of non-reversible photoinhibition and photosynthetic apparatus damage. These two sequential phases have been described in the response to severe DS in other woody and perennial crops , the results provided insights indicating that the clones presented differences in their responses related to their capacity to tolerate DS, at least under our experimental conditions. Thus, our results indicate that the EET8 clone was the most performant under DS, whereas ICS60 was seemingly the less tolerant. These differences could reflect changes in acclimation responses, which in turn could be either genetically or epigenetically dependent .On the other hand, as photoinhibition mechanisms were observed under DS, which suggests secondary oxidative stress induction, a preliminary approach to the activation of the antioxidant system at the transcriptional level was carried out in these clones to complement these findings.Besides, considering that grafting practice is frequently used to integrate traits of interest between the rootstock and the scion, which in cacao is mainly destinated to confer disease resistance , and thaGST and HSP showed high induction in the three clones evaluated at D23, which likely had a much earlier induction. This finding could be related to an effort of cacao plants to prevent or attenuate the negative effects of ROS increase triggered by the DS imposed. This activation has been reported as an early response induced in plants to protect protein structures and lipids against possible damage caused by ROS and tends to accumulate as a consequence of photorespiration  reactions were affected and that a typical oxidative stress response occurred in cacao upon DS. Indeed, gene activation related to oxidative stress-related genes was seemingly directed to protect leaf cells because the expression of canonical oxidative stress repair marker genes piration .v/Fm, which likely affected A, and the drop in gs, with both responses evidenced during DS. GST is a key enzyme in ROS detoxification due to its conjugation with glutathione  may indicate that the transcriptional activation of this pathway in T. cacao could have happened earlier, even before D23, when the water status-related physiological parameters started to decrease and the related stress was perceived systemically by the plant. Moreover, this early upregulation could also be related to the physiological regulation of the stress response since the RBOHF protein exerts regulatory functions on ABA, the master mediator of the systemic response to DS , suggesting probable coregulation as part of the common gene regulatory network controlling this systemic response to DS. Indeed, thioredoxins (NTRCs) are disulfide reductases that regulate the redox state of other proteins and lipid peroxides. Therefore, their function has been related to the maintenance of metabolic pathways occurring in chloroplasts, such as photosynthesis, because of stress conditions  gene  gene . Rehman 2 supply or by mitochondrial damage  or complete depletion (anoxia), the cytoplasmic pH drops, increasing the activity of the two fermentation enzymes (pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH)) responsible for ethanol accumulation in the cytoplasm of cells. This ethanol allows the regeneration of NAD+ needed for glycolysis to continue producing the energy (ATP). In addition, it contributes to maintaining membrane integrity and its minimal functioning and stabilizing cellular pH, thereby avoiding lethal acidosis until aerobic respiration resumes  , which could be a possible explanation for the general downregulation pattern observed at the transcriptional level for some of the fermentative metabolism genes, as well as all genes evaluated, and caused by a drastic change in energetic metabolism, with a probable trade-off between fermentative metabolism and maintenance respiration. However, transcriptional repression of a gene does not necessarily imply a drop in its respective protein level or activity, which could be maintained further. A similar behavior was described in conifers, which showed that all aerial tissues of drought-stressed seedlings accumulated ethanol when the tissues were near death or dying  linked to the stress-avoidance mechanism; then, when DS became severe, a second phase response emerged with the downregulation of photosynthetic efficiency  as a consequence of oxidative stress and photoinhibition caused by DS. However, all plants recovered quickly (4 DAR), probably by mediating the antioxidant system as well as the efficiency of PSII repair mechanisms and the turnover of chloroplasts and their components, which represent crucial responses for maintaining photosynthesis and metabolism under stress conditions . AlthougThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.MO, LR, and WT conceived the project and experimental design. DC designed the gene expression profiling. MO and DC carried out the experiments, collected, analyzed data, and wrote the initial versions of the manuscript. All authors contributed to manuscript revision and improvement, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Test meals included breakfast, lunch, snack and dinner and the daily GL was reduced by 37% in the LGL diet. Continuous glucose monitoring provided 24 h glycemic excursion and variability parameters: incremental area under the curve (iAUC), max glucose concentration (Max), max glucose range, glucose standard deviation (SD), and mean amplitude of glycemic excursion (MAGE), time in range (TIR). Over 24h, the LGL diet resulted in a decrease in glucose Max , glucose range , glucose SD , mean amplitude of glycemic excursion , and increase in 4.5–6.5mmol/L TIR , compared to the HGL diet. The glucose iAUC, MAX, range and SD improved during the 2 h post-prandial window of each LGL meal, and this effect was more pronounced later in the day. The current results validate the dietary strategy of incorporating innovative functional ingredients  and replacing Asian staples with alternative low GI carbohydrate sources to reduce daily glycemic load to improve glycemic control and variability as a viable alternative to the reduction in carbohydrate intake alone. These observations provide substantial public health support to encourage the consumption of staples of low GI/GL to reduce glucose levels and glycemic variability. Furthermore, there is growing evidence that the role of chrononutrition, as reported in this paper, requires further examination and should be considered as an important addition to the understanding of glucose homeostasis variation throughout the day.A reduction in carbohydrate intake and low-carbohydrate diets are often advocated to prevent and manage diabetes. However, limiting or eliminating carbohydrates may not be a long-term sustainable and maintainable approach for everyone. Alternatively, diet strategies to modulate glycemia can focus on the glycemic index (GI) of foods and glycemic load (GL) of meals. To assess the effect of a reduction in glycemic load of a 24 h diet by incorporating innovative functional ingredients  and alternative low GI Asian staples on glycemic control and variability, twelve Chinese men (Age: 27.0 ± 5.1 years; BMI:21.6 ± 1.8kg/m The prevalence of type 2 diabetes and related risk factors have increased dramatically across Asia in recent decades and Singapore is no exception. Improved economic prosperity, advances in technology, increased food production, availability and safety have allowed a shift towards a lifestyle with reduced physical activity and a diet with a greater overall carbohydrate intake, lower fiber and higher sugar and fat content . In addiChronic high blood glucose levels are considered a major risk factor for the onset of diabetes; in addition, greater glucose variability has independently been linked to the increased risk for the development of diabetes and cardiovascular diseases (CVD) ,5,6, likDiet strategies to manage glycemia have included frequent small meals  and reduAs part of dietary strategies, several functional food ingredients have been shown to moderate the postprandial glycemic response. Dietary fibers refer to plant components that are predominantly non-digestible by the human digestive system. β-glucan, a soluble fiber, which is a glucose polymer, can reduce glucose availability and its inclusion in foods can lower the postprandial glycemic response and the GI ,25,26,27To enhance the adoption of healthier foods of low GI by consumers and the food industry, it is necessary to develop and offer these foods based on staples consumed in Asian countries. The current study used alternative low GI Asian staples and healthier food prototypes and were specifically formulated based on Singapore’s Healthier Choice nutrient guidelines and included in this study for physiological validation . In this2, had fasting blood glucose below 6 mmol/L and had a blood pressure below 140/90 mm·Hg. Exclusion criteria were metabolic diseases , known glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency), medical conditions and/or taking medications known to affect glycemia , intolerances or allergies to test foods, sports participation at a competitive and/or endurance levels, intentional food intake restriction and smoking. One drop-out and two incomplete data sets resulted in a total of 12 included data sets for analysis.Fifteen healthy Chinese male subjects between 21 and 40 years were recruited by a variety of methods including flyers, online advertisement and the research centre’s volunteer database. Volunteers had a body mass index (BMI) between 18.5 to 25 kg/mThe study was conducted at the Clinical Nutrition Research Centre (CNRC), Singapore, in accordance with the guidelines laid down in the Declaration of Helsinki. All procedures involving human participants were approved by the Domain Specific Review Board (DSRB) of the National Healthcare Group, Singapore (Reference no. 2017/00994). The protocol was well explained to the volunteers and written informed consent was obtained from each subject before participation. This study was registered in the clinicaltrial.gov registry as NCT03703544.http://www.randomizer.org/ (accessed on 9 August 2021)) [In a randomized, single-blind, controlled, cross-over design, subjects attended two test sessions separated by a wash-out period of at least 3 days. The two test sessions included either a reduced glycemic load (LGL) or a high glycemic load (HGL) 24 h meal sequence . The 24 h meal sequence was based on typical Asian foods and included innovative functional ingredients  and alternative low GI Asian staples . The macronutrient and energy composition of the meals is provided in t 2021)) .The two test sessions included either a reduced glycemic load (LGL) or a high glycemic load (HGL) 24 h meal sequence . The two diets had an equal amount of available carbohydrates and were closely matched in energy content and macronutrient distribution . The glyContinuous glucose monitoring (CGM)  was used to measure glycemic response, defined as the primary outcome. The insertion was performed on Day 1 at approximately 16:00 and the sensor was removed on Day 3 of the study at 09:00. Data was collated and processed using online software . The data reported in this paper represent 24 h interstitial glucose readings recorded every 5 min from the start of breakfast on Day 2 at approximately 09:00 until 24 h later at approximately 09:00 on Day 3. During each test session, the CGM sensor was calibrated against finger-stick blood glucose measurements four times a day before every meal and before sleeping using the FreeStyle Optium Neo Blood Glucose meter (Abbott Laboratories). A cross-over design with a minimum of 8 subjects would be sufficient to detect a 15% change in area under the glucose curve (24 h) with a power of 0.85 at a significance level of 0.05 .p < 0.05.All statistical analyses were performed using Statistical Package for the Social Sciences version 23 . Values are presented as mean ± SEM unless otherwise stated. Prior to statistical analysis, the normality of the data was assured using the Shapiro-Wilks test. Statistical significance was set at www.easygv.co.uk, accessed on 9 August 2021) [The primary outcome was to determine how a 24 h meal sequence based on a LGL diet using functional ingredients and healthier alternative food staples impacts the 24h glycemic response and variability. Firstly, the baseline glucose value for each subject was determined from the 1h average CGM interstitial glucose readings in a fasted state from 06:00 to 07:00 on day 2 and used to calculate the change in glucose level for the 24 h. Glycemic response was expressed as the incremental area under the curve (iAUC), calculated using the trapezoidal approximation method and the change in glucose above baseline (baseline-corrected) . The totst 2021) . All parAnthropometric measurements and characteristics of the subjects can be found in The cumulative glycemic load of the LGL was 37% lower compared to the HGL diet (165 vs. 263) and the reduction was fairly consistent among the four meals: −31% for breakfast, −43% for lunch, −36% for snack, and −34% for dinner . This eqp = 0.448) over 24 h between the LGL and HGL diet. Parameters indicative of glycemic variability showed that over 24 h, the LGL diet resulted in a decrease in: glucose Max , glucose range , glucose SD , mean amplitude of glycemic excursion , and increase in 4.5–6.5mmol/L TIR (+15% p = 0.009), compared to the HGL diet. In summary, the current data suggests a great improvement in GV over 24h following the LGL diet.The incremental change in glucose compared to baseline is depicted in p = 0.0015), glucose Max , glucose range , glucose SD  compared to the HGL dinner. Comparing the improvements per meal relative to their reduction in GL  between the meals showed an IF of 1.27 for breakfast, 1.27 for lunch, 1.62 for the snack and 2.28 for the dinner respectively. In other words, each percentage point reduction in GL during dinner, reduced the iAUC with 2.28% compared to only 1.27% during breakfast and lunch.Meal-specific analysis revealed an improvement in almost all determined glycemic parameters for each of the meals . The greThe 24 h reduced GL diet resulted in a much improved 24 h glycemic response compared to the control high GL diet as indicated by: reduced glycemic variability, lower peak glucose levels, greater time in range, and improved post-prandial glycemic response after each meal. The “healthier choice” diet with innovative ingredients improved glycemic variability and glycemic control, two major independent risk factors in the development and progression of type 2 diabetes. Therefore, this study validates the use of low GI foods to reduce GL as an alternative dietary strategy to limiting carbohydrate intake alone in Asians to manage glycemia. The reduction in GL was accomplished by incorporating innovative functional ingredients  and replacing Asian staples with alternative low GI carbohydrate sources to improve carbohydrate quality. Notably, a relatively greater beneficial effect on glucose control was observed during the evening compared to the morning.Consistently, studies have demonstrated that oat and barley products which are natural sources of β-glucan and starchy products fortified with β-glucan aid in reducing glycemic response without an increase in insulin secretion to mediate increased glucose clearance . Likely Isomaltulose has a low GI and studies have repeatedly shown a decreased glycemic response compared to sucrose without an increase in insulin as the underlying mechanism ,46,47. HIn the current study an overall greater relative reduction in post-prandial glycemic response was observed in the evening following a reduced GL diet. The results showed that consumption of the LGL versus HGL diet at dinner, resulted in a greater relative reduction in the glucose iAUC, maximum value, range and variability (SD and MAGE), compared to the decrease earlier on the day following the LGL breakfast and lunch. Our study was not specifically designed to assess the effect of mealtime on glucose response, as the meals for breakfast, lunch and dinner were not identical. Nonetheless, this important observation on mealtime effect may be of clinical relevance. In addition, the relative reduction in GL between the HGL and LGL meals was consistent. Furthermore, when the relative improvement in iAUC was adjusted for the relative decrease in GL for each meal, the results indicated that for each 1% reduction in GL, the iAUC was reduced with 2.28% percentage point during dinner compared to only 1.27% during breakfast and lunch.Our findings are in line with Morgan et al. who found that a low GI diet lowered the post-prandial glucose response both in the morning and the evening, though to a greater extent in the evening . FurtherOur study further illustrates the limited use and application of single point measurements or average glucose values over a 24 h period ,13,14. TA minor limitation was the male-only study population, though similar results can be expected in women as previous studies have not demonstrated gender differences in mixed study populations ,58. The The current results validated the glycemic load reduction strategy by replacing staples with “healthier choice” options containing innovative functional ingredients  and replacing Asian staples with alternative low GI carbohydrate sources. Functional ingredients can improve the carbohydrate quality and the inclusion of such ingredients is a potential way to improve postprandial glycemia and glycemic variability. These observations provide substantial public health support to encourage the consumption of staples of greater carbohydrate quality as a viable alternative to the reduction in carbohydrate intake alone for glycemic control. Increased opportunities for research and development of highly palatable, low GI, fortified Asian food staples is required to aid in the prevention and treatment of diabetes among Asians. In addition, there is growing evidence that the role of chrononutrition requires further examination and should be addressed in the light of practical nutrition recommendations.


Traditional Chinese Medicine (TCM) is distinguished by Syndrome differentiation, which prescribes various formulae for different Syndromes of same disease. This study aims to investigate the underlying mechanism.Using a strategy which integrated proteomics, metabolomics study for clinic samples and network pharmacology for six classic TCM formulae, we systemically explored the biological basis of TCM Syndrome differentiation for two typical Syndromes of CHD: Cold Congealing and Qi Stagnation (CCQS), and Qi Stagnation and Blood Stasis (QSBS).Our study revealed that CHD patients with CCQS Syndrome were characterized with alteration in pantothenate and CoA biosynthesis, while more extensively altered pathways including D-glutamine and D-glutamate metabolism; alanine, aspartate and glutamate metabolism, and glyoxylate and dicarboxylate metabolism, were present in QSBS patients. Furthermore, our results suggested that the down-expressed PON1 and ADIPOQ might be potential biomarkers for CCQS Syndrome, while the down-expressed APOE and APOA1 for QSBS Syndrome in CHD patients. In addition, network pharmacology and integrated analysis indicated possible comorbidity differences between the two Syndromes, that is, CCQS or QSBS Syndrome was strongly linked to diabetes or ischemic stroke, respectively, which is consistent with the complication disparity between the enrolled patients with two different Syndromes. These results confirmed our assumption that the molecules and biological processes regulated by the Syndrome-specific formulae could be associated with dysfunctional objects caused by the Syndrome of the disease.This study provided evidence-based strategy for exploring the biological basis of Syndrome differentiation in TCM, which sheds light on the translation of TCM theory in the practice of precision medicine.The online version contains supplementary material available at 10.1186/s13020-021-00521-3. 1. Our work was based on clinical samples rather than pure data analysis or animal models.2. We conducted multiple omics studies. Especially, as for metabolomics study, we performed both untargeted and targeted metabolomics experiments.3. We performed network pharmacological study to cross-validated the results of multi-omics study. Although the data sources of network pharmacology were completely unrelated with our omics data, they came to the same conclusion about the difference of the two Syndromes.4. In the network pharmacological study, we made efforts to collect and screen high-quality data. We collected data from multiple TCM databases and conducted drug likeness screening. Especially, we added quality markers of each herb, whose pharmacological relevance had been validated. To enhance the reliability of targets, for each Syndrome, we only studied common targets of 3 different TCM formulae prescribed for this Syndrome.The online version contains supplementary material available at 10.1186/s13020-021-00521-3. Traditional Chinese medicine (TCM) has developed over thousands of years in China, in which the medication is mainly practiced in the form of TCM formula based on Syndrome (ZHENG in Chinese) differentiation by TCM physicians , 2. SyndCoronary heart disease (CHD) lists the first position in mortality in the world, which includes four classes, i.e. nonobstructive coronary atherosclerosis, unstable angina pectoris, stable angina pectoris, and acute myocardial infarction . MeanwhiIn our current study, an integrated multi-omics and network pharmacology strategy was proposed for elucidating the scientific basis of patient stratification of TCM Syndrome in a group of patients with CHD who were also diagnosed as either CCQS or QSBS Syndrome by TCM physicians. Between August 2018 and December 2019, we collected serum samples of 111 participants, including 44 CHD patients with CCQS Syndrome, 37 with QSBS Syndrome and 30 healthy people from the Cardiology Clinic of Dongzhimen Hospital affiliated to Beijing University of Chinese Medicine and the neighboring communities in Beijing. The blood samples were then used in metabolomics and proteomics studies. Pathways involved in the pathologic process of the two Syndromes were discovered by functional annotation of differential proteins and metabolites. For each of the two Syndromes, we selected 3 classic TCM formulae clinically used for the treatment of the Syndrome. The common putative targets of the 3 formulae were considered as feature targets of the Syndrome and used for network pharmacological study and integrated analysis with omics data. At last, we identified key biological processes and molecules that may associated with the occurrence and development of the two Syndromes of CHD.The workflow of the study is illustrated in Fig. This study was conducted in compliance with the Declaration of Helsinki and the requirements of clinical trials by the Drug Administration Law of the People's Republic of China in Dongzhimen Hospital, Beijing between August 2018 and December 2019. The protocol and informed consent were reviewed and approved by the Beijing University of Chinese Medicine Clinical Trials and Biomedical Ethics Committee (No. DZMEC-KY-2019–121). Informed consent was obtained from all participants and their privacy rights were always preserved. All patients were enrolled by the Cardiology Clinic of Beijing University of Chinese Medicine or the surrounding communities in Beijing. Whether it meets the inclusion criteria (CCQS and QSBS Syndrome of CHD) is determined by an expert , with verification by two assistants (Yuanyuan Zeng and Jing Zhao).Diagnostic criteria for CHD referred to the “Diagnostic Criteria for Coronary Heart Disease” defined by the International Society of Cardiology and Association and the World Health Organization in 1979: (1) With a history of typical angina pectoris or myocardial infarction, except for valvular heart disease, coronary embolism and cardiomyopathy; (2) Patients over the age of 40 for men and over 45 for women, the ECG myocardial ischemia in resting state, or the treadmill exercise test is positive, without other reasons ; (3) Among the risk factors of coronary heart disease, two of the three items of hypertension, diabetes and hyperlipidemia can be clinically diagnosed for coronary heart disease. Or coronary angiography or coronary CT scan shows that at least one main branch vessel has a stenosis greater than 50% to confirm the diagnosis of coronary heart disease. In this study we mainly selected patients with stable angina while satisfying the diagnosis of coronary heart disease. They are characterized by paroxysmal squeezing pain or suffocation in the anterior chest, mainly located at the back of the sternum, which can radiate to the anterior heart area and left upper limb. The ulnar side usually occurs when the labor load increases and lasts for a few minutes. The pain disappears after resting or using nitrate ester preparations. The degree, frequency, nature, and predisposing factors of pain episodes did not change significantly within weeks to months.According to the “Guiding Principles for Clinical Research of New Chinese Medicines”, the diagnostic criteria for Syndromes were determined as follows:Diagnostic criteria for CCQS Syndrome: primary symptoms include chest tightness, chest pain. First set of secondary symptoms includes the condition or pain aggravates on rainy days or cold, face pale, like warmth and fear of cold, cold limbs, cramps and pain. Second set of secondary symptoms includes heart palpitations, fullness of the chest and flanks, fullness of the abdominal abdomen. The tongue is pale or dark, and the tongue coating is white. Late or tight pulse.Diagnostic criteria for QSBS Syndrome: primary symptoms include chest tightness, chest pain. First set of secondary symptoms includes that the condition is related to emotions, emotional depression, irritability, sigh, and chest fullness; The second set of secondary symptoms includes palpitations, rough, dry, and hyperkeratotic skin, sublingual varicose veins, dark purple face and lips, dark purple tongue or petechiae, and astringent pulse.Those who have one of the primary symptoms, one or more of the first and second set of secondary symptoms at the same time, and the tongue and pulse conditions are consistent, can be diagnosed. In the diagnosis process, three researchers  independently differentiated syndromes at the same time and then determined the types of syndromes together.Patients meeting the diagnostic criteria were potentially eligible for the study if they meet the following criteria: (1) age at 18 to 85 years old; (2) clearly diagnosed as angina pectoris, and are classified as CCQS or QSBS in the chest pain of TCM after the doctor's differentiation witness; (3) the included patients have stable vital signs, clear consciousness, and certain expression skills; (4) voluntary submission of written informed consent prior to enrollment.Patients would be excluded if they meet one of the following criteria: (1) those who have had myocardial infarction within 3 months or have undergone coronary revascularization; (2) patients with other serious heart diseases: such as valvular heart disease, hypertrophic cardiomyopathy, severe arrhythmia , severe cardiac insufficiency; (3) patients with severe liver and kidney dysfunction; (4) pregnant and lactating women; (5) those who use traditional Chinese medicine decoctions and related preparations within 2 weeks; (6) those who have participated in other drug clinical trials within 2 weeks.4HCO3 solution and digested with trypsin . Peptides were collected with 10 kDa filter .Serum proteins were prepared by in solution digestion method. All samples were randomly divided into 11 experimental groups. A standard sample was added to each experimental group and prepared at the same time. We processed one set of samples (including a standard sample) per day and all samples and standard samples were prepared within 11 days. In brief, lysis solution was added into the collected human serum samples. Then DTT and IAA were added. After this procedure, we added precipitation buffer and placed it in -20 ℃ refrigerator overnight. The cold concentrate containing proteins was centrifugated and then removed the supernatant. Pre-cooled 100% acetone was added into the precipitated proteins and removed by centrifugating. Then precooling 70% ethanol was added into the proteins and the procedure was same as before. The above two steps were repeated twice. Proteins were resuspended with 100 mM NHEqual volume of all human serum samples was pooled for the generation of a spectral library. The pooled sample was depleted by High-Select™ Top14 Abundant Protein Depletion Resin (Thermo Fisher). The sample with abundant proteins removed was further digested by trypsin with the in-solution digestion method. Digested peptides were desalted and fractionated through the high-pH reverse phase liquid chromatography using a Waters XBridge BEH300 C18 3.5 μm 2.1 × 150 mm column on Agilent 1200 LC instrument using an 85-min gradient.EASYnLC 1000 HPLC system (Thermo Fisher Scientific) and Q Exactive HF mass spectrometer (Thermo Fisher Scientific) were used for LC–MS/MS analysis. The collected peptides were separated on a home-made column . Each fractionation of peptides for spectral library generation was analyzed with data dependent acquisition (DDA) mode and separated with a 120-min gradient. For DIA analysis, desalted peptides concentration was measured by NanoDrop 2000 (Thermo Scientific). Then peptides were separated on a 70-min LC gradient. The DIA acquisition scheme consisted of 32 variable windows ranging from 350 to 1600 m/z.http://maxquant.org/, version 1.6.7.0). The results were imported into Spectronaut 14.0 (Biognosys) for library generation. And the generated library contained 1,095 proteins and 11,871 precursors. For human serum, DIA data was processed with Spectronaut 14.0. Statistical analysis was performed in R software. Significantly differential proteins were determined using Student’s t-test with a p-value < 0.05 and fold change (FC) > 1.1.DDA data for spectral library generation were analyzed via MaxQuant software (v/v) precooled to − 20 ℃. After vortex mixed for 30 s and sonication for 10 min in an ice bath, samples were overnight at − 20 ℃ to improve protein precipitation and then centrifuged at 12 000×g for 15 min at 4 ℃, 2 µl of supernatant was subjected to HPLC-QTOF-MS/MS analysis.An aliquot of 50 µl of thawed serum sample was deproteinized with 150 µl of MeOH: ACN  and ACQUITY UPLC BEH C18 column ] were used for untargeted metabolomics analysis.Sample analysis was performed on a Shimadzu Nexera XR LC-20AD HPLC system equipment with SCIEX Triple TOF 5600https://hmdb.ca/), LIPIDMAPS (https://www.lipidmaps.org/), KEGG (https://www.kegg.jp/) and METLIN (https://metlin.scripps.edu) were used to align the molecular mass data to identify metabolites.The raw data were imported to the Progenesis QI for peak alignment to obtain the peak area list and the identification result list. The nonparametric univariate method (Mann–Whitney-Wilcoxon test) was used to analysis metabolites that differed in abundance between the different subgroups corrected for false discovery rate (FDR) to ensure that the peak of each metabolite was reproducibly detected in the samples. Metabolites selected as biomarker candidates for further statistical analysis were identified on the basis of variable importance in the projection (VIP) threshold of 1 from the tenfold cross-validated OPLS-DA model, which was validated at a univariate level with FDR < 0.05. The online HDMB data (g for 30 min at 4 ℃. The supernatant (30 µl) was transferred into a new 96-well plate containing 20 µl of derivatization reagent, and the mixture was reacted at 30 ℃ for 60 min. After reaction, we added 350 µl 50% methanol solution precooled to -20 ℃ for 20 min, then centrifuged at 4000 × g for 30 min at 4 ℃, the supernatant (135 µl) was transferred into a new 96-well plate containing 15 µl of IS. Gradient dilutions of the derivatized standard stock solution were added to the left hole, and finally the plate was sealed for LC–MS analysis.An aliquot of 25 µl of thawed serum sample was added to the pre-chilled 96-well plate, then added 100 µl of methanol containing internal standard (IS) vortex for 5 min, and finally centrifuged at 4000×Chromatographic analysis was performed using a Waters ACQUITY I-Class UPLC equipped with an ACQUTIY UPLC BEH C18 column . Waters Xevo TQ-S triple quadrupole mass spectrometer was combined with UPLC system via the electro-spray ionization (ESI) source in both positive and negative ionization modes. We performed accurate quantitative analysis of 306 metabolites  in this study.The targeted raw data were processed through calibration curve of standards. Then, the raw data were analyzed by the iMAP software . Metabolites selected as biomarker candidates for further statistical analysis were identified on the basis of variable importance in the projection (VIP) threshold of 1.0 from the tenfold cross-validated OPLS-DA model, which was validated at a univariate level with p value < 0.05.More detailed process descriptions of proteomics and metabolomics are provided in Additional file For each of the two TCM Syndrome of CHD, we selected 3 formulae applied in clinic for the treatment of this Syndrome. The treatment of each formula to corresponding Syndrome was recommended in the Expert Consensus for Chinese Medicine Diagnosis and Treatment of Stable Angina Pectoris of CHD  and alsoProtein–protein interaction networks were construction by GeneMania platform . MetabolFunctional annotation analysis for genes was conducted by DAVID  and STRIn = 44), QSBS (n = 37) and healthy controls  were enrolled at the Cardiology Clinic of Dongzhimen Hospital affiliated to Beijing University of Chinese Medicine or the surrounding communities in Beijing between August 2018 and December 2019. The demographic and clinical biochemical indicators of the participants were listed in Table A total of 111 participants, including CHD patients with CCQS Syndrome , and TG < 1.7 mmol/L (GPO-POD method). This bias could be due to the higher age of participants (most between 60 and 70), meanwhile participants in CHD group may have been given some lipid-lowering interventions.In order to study the proteomic characteristics, we used the data independent acquisition (DIA) method to analyze the serum samples. In total, 470 proteins were identified. Averagely, 353, 355 and 354 proteins were identified in CCQS, QSBS and HC group, respectively Fig. A. We alsNext, to figure out the proteomic profile differences between CCQS and QSBS Syndrome, 388 proteins  were included in subsequent analysis. Differential proteins (DPs) between CCQS and HC groups, as well as QSBS and HC groups  were determined. There were 24 differential proteins between CCQS and HC groups, with 5 up-regulated and 19 down-regulated , respectively. Table To explore the metabolic differences between CCQS and QSBS Syndromes, we constructed a partial least squares discriminant analysis (PLS-DA) model. The serum metabolome of CCQS and QSBS patients were clearly separated from health control on the PLS-DA score plot in both untargeted Fig. A and tarOn the basis of identified DMs, a total of 91 and 97 DMs with KEGG IDs were further analyzed, which were significantly altered in CHD patients of either CCQS or QSBS Syndrome, respectively. Then, we used MetaboAnalyst platform to perform functional enrichment analysis for the DMs. Compared with healthy controls, seven differential metabolic pathways were enriched in the serum of CCQS patients, including lipid metabolism , amino acid metabolism , metabolism of cofactors and vitamins (pantothenate and CoA biosynthesis) Fig. E. Compari.e., glycerophospholipid metabolism, and sphingolipid metabolism; while DMs of QSBS were enriched in 4 pathways of amino acid metabolism, including arginine metabolism, tyrosine metabolism, and two pathways of glutamate metabolism.Figure l-valine and pantetheine), 2 amines (sphinganine and ethanolamine), were unique metabolites in the serum of patients with CCQS. The 15 DMs, including 10 amino acids , 2 fatty acyls , 1 glycerophospholipids (PC (20:1/14:1)), 1 benzenoids (phenylpyruvic acid), and 1 organic acid (oxoglutaric acid), were unique metabolites in the serum of patients with QSBS.Next, we mapped the network of differential metabolic pathways (p < 0.05) enriched with DMs through Cytoscape software. As shown in Fig. l-serine, 5-aminolevulinic acid, arachidonic acid, linoleic acid, PC(16:0/16:0), oxalic acid, oxoglutaric acid) and 6 proteins  form the QSBS network. Three fatty acids metabolites  and 1 protein (APOB) are the same metabolites and protein included in the two networks. Arachidonic acid, linoleic acid, eicosapentaenoic acid are fatty acids metabolites significantly associated with inflammation, which suggests that inflammation may be a common feature of the two Syndromes. Figure First, we used MetaboAnalyst platform to study the interactions between metabolites and proteins associated with the same Syndrome. We input DMs and DPs of the same Syndrome into the “Network Analysis” module of MetaboAnalyst and constructed the integrated metabolite-protein network of Syndrome. As shown in Fig. l-valine, pantetheine, lysoPC (20:2), sphingosine 1-phosphate) and , socitric acid), could accurately distinguish CCQS and QSBS patients from healthy controls, AUC were 0.84 and 0.81, respectively  could accurately distinguish CCQS patients from healthy controls. as indicated by the area under the receiver operating curve (AUC), which had a value up to 0.844 Fig. D. Howeveely Fig. F, G. Theely Fig. F, G.To investigate features of the two distinct Syndromes from TCM formulae specifically used to treat them, we selected 6 TCM formulae recorded in the Chinese Pharmacopeia and extensively used for the treatment of the two TCM Syndromes of CHD in clinic. Each Syndrome corresponded to 3 formulae. The formulae prescribed for CCQS Syndrome were Shexiang Baoxin Pill, Guanxin Suhe Pill, and Kuanxiong Aerosol; while those for QSBS Syndrome were Suxiao Jiuxin Pill, Xuefu Zhuyu Pill, and Compound Danshen Drop Pill  were also identified by integrated analysis. One pathway specific for CCQS (pantothenate and CoA biosynthesis) were identified both in metabolomics and integrated analysis. Three pathways specific for QSBS  identified by metabolomics analysis, were also identified here.We then applied MetaboAnalyst platform for functional annotation of the two metabolite-gene networks. We focused the analysis on the basic biological pathways of KEGG database, which contains 5 sections: Metabolism, Genetic Information Processing, Environmental Information Processing, Cellular Processes, and Organismal Systems. All the molecules, including DMs, DPs and feature targets, in the metabolite-gene network of CCQS were input into the “Joint-Pathway Analysis” module of MetaboAnalyst and 86 pathways enriched with molecules related with CCQS were identified (p < 0.05). Similarly, 80 pathways were found to be enriched with molecules associated with QSBS (p < 0.05). We conducted intersection analysis for the two groups of pathways and discovered that the two Syndromes shared 66 pathways, in which the top 35 pathways were show in Fig. Next, we applied DAVID platform to identify disease groups enriched with Syndrome-specific genes included in the integrated networks. We input the 2 DPs and 17 feature targets specific for CCQS Syndrome in the metabolite-gene network into DAVID and performed functional annotation clustering with classification stringency “High”. QSBS-specific genes in the metabolite-gene network were analyzed in the same way. Table In this study, we proposed a strategy which integrated clinic samples, multi-omics study, and network pharmacology to investigate the biomedical basis of two Syndromes (CCQS and QSBS) of CHD.We collected clinic samples and conducted proteomic and metabolomic experiments using serum samples of the participants. Statistics suggested that clinic samples of the two Syndromes had no differences in age, sex, BMI and laboratory indexes, but the comorbidity features were different. CCQS group included higher percentage of patients with diabetes, whereas more patients in QSBS group had complications of cerebrovascular disease. Our functional annotation clustering for proteomics and metabolomics results also reflected the comorbidity difference in clinic samples of the two Syndromes. Furthermore, network pharmacological study on 6 TCM formulae used for the treatment of two Syndromes revealed that the 3 CCQS formulae paid more attention on the treatment for diabetes complication of CHD, while the 3 QSBS formulae focused more on the management of ischemic stroke complication related to CHD. This result suggested that the comorbidity discrepancy between the two Syndromes might be due to fundamental differences in the two Syndromes, rather than a deviation in sample collection. Further research and more evidence are needed for confirming this observation about comorbidity.By analyzing differential proteins (DPs) of the two Syndromes identified from proteomic experiments of clinic serum samples, it was found that DPs of both Syndromes were enriched in the pathways of cholesterol metabolism, PPAR signaling, vitamin digestion and absorption, and fat digestion and absorption, suggesting that these 4 pathways were involved in the disease process of both Syndromes. It has been recognized for decades that CHD is strongly related with endogenous or exogenous lipids. Cholesterol is made by the body through the digestion and absorption of dietary fat, and plasma LDL cholesterol levels have an independent correlation to the risk of CHD . PPARs  of both Syndromes were enriched in 4 pathways: linoleic acid metabolism; biosynthesis of unsaturated fatty acids; glycine, serine and threonine metabolism; and cysteine and methionine metabolism, suggesting that the 4 pathways were associated with both Syndromes of CHD. It has been known that dietary unsaturated fat intake is inversely correlated with CHD risk . LinoleiAt last, by integrating data from network pharmacology, proteomics, and metabolomics study, we constructed integrated metabolite-gene network for the two Syndromes and conducted functional annotation for molecules in the two networks, respectively. The results of integrated analysis and omics (metabolomics and proteomics) analysis were consistency in 5 common pathways for two Syndromes , 1 specific pathway for CCQS (pantothenate and CoA biosynthesis), and 3 specific pathways for QSBS . Disease clustering analysis for Syndrome-specific genes in the integrated networks suggested that the genes of both Syndromes were enriched in coronary atherosclerosis. In addition, CCQS-specific and QSBS-specific genes were also enriched in metabolic Syndrome and cerebrovascular disease, respectively, in which Syndrome-specific DPs involved in the diseases were PON1 and ADIPOQ for CCQS, APOE and APOA1 for QSBS. According to our proteomics experiments, the abundance of PON1 and ADIPOQ were significantly lower in CCQS patients compared with healthy control, while both APOE and APOA1 were down-expressed in QSBS patients compared with healthy control, suggesting that the two pairs of DPs could play important roles in the pathology of the two Syndromes.PON1 (serum paraoxonase/arylesterase 1) is one of the 3 members of the paraoxonase family, any of which can degrade lipid peroxides in HDL and LDL . PrimariADIPOQ (adiponectin) is an adipokine produced by adipose tissue that regulates lipid metabolism and insulin sensitivity. In vitro experiments have shown that ADIPOQ suppressed the expression of endothelial adhesion molecules, the production of TNFa and IL6, the proliferation of vascular smooth muscle cells, and the transformation of macrophage to foam cells , 46. LowAPOE (apolipoprotein E) is an apolipoprotein important for the transport and metabolism of lipids. It is abundant in the brain . APOE coAPOA1 (apolipoprotein A-I) is the major protein component of high-density lipoprotein (HDL) complexes. By boosting cholesterol efflux from tissues, it plays a key function in the reverse transfer of cholesterol from tissues to the liver for excretion. The quantity of ApoA1 in plasma has been found to be substantially associated with the level of HDL cholesterol in the blood . AccordiTo sum up, based on clinic samples and collected data, our study revealed the differences between the two Syndromes from three aspects: biological processes, comorbidities, and potential biomarkers. We should point out some limitations in this research. The first is that the size of clinic samples is relatively small due to the limitation of time period, as well as strict inclusion and exclusion criteria. Second, formulae’s ingredients and targets are collected from databases. Although we integrated our data from most TCM databases currently available in public and studied multiple TCM formulae to enhance the reliability of targets, the inherent quality of the databases themselves may cause some deviation in our results.By integrating clinic samples, multi-omics study and network pharmacology, our study suggested that both CCQS and QSBS Syndrome of CHD were related with the dysfunction of 5 pathways: glycine, serine and threonine metabolism; biosynthesis of unsaturated fatty acids; linoleic acid metabolism; cholesterol metabolism; and PPAR signaling pathway. Moreover, CCQS Syndrome of CHD patients were specifically characterized with altered pantothenate and CoA biosynthesis, while disordered pathways of D-glutamine and D-glutamate metabolism; alanine, aspartate and glutamate metabolism, and glyoxylate and dicarboxylate metabolism were present in CHD patients with QSBS Syndrome. Furthermore, our results indicated that the down-expressed PON1 and ADIPOQ could related with the pathology of CCQS Syndrome, while down-expressed APOE and APOA1 for QSBS Syndrome. In addition, both clinic samples and network pharmacological results suggested that CCQS or QSBS Syndrome was highly related with the pathology of diabetes or ischemic stroke, respectively. Further research needs to be conducted to investigate whether such comorbidity phenomena is correlated with the inherent difference of the two Syndromes. This study helps us to understand the TCM Syndromes from perspective of modern biomedical science and facilitate the application of TCM theory in the practice of precision medicine.Additional file 1. Materials and methods for proteomics and metabolomics experiments. Table S1. List of differential metabolites. Table S2. Herbs in the 6 TCM formulae under study. Table S3. Active compounds in each formula. Table S4. Putative targets in each formula. Table S5. Diseases the most highly associated with CHD and corresponding drug ATC codes.