However, the mechanisms of DNA condensationare not fully understood due to the inability of experiments to accesscation distributions and the complex interplay of energetic and entropicforces during assembly. By constructing free energy surfaces usingexhaustive sampling and detailed analysis of cation distributions,we elucidate the mechanism of DNA condensation in different salt conditionsand with different DNA sequences. We found that DNA condensation isfacilitated by the correlated dynamics of the localized cations atthe grooves of DNA helices. These dynamics are strongly dependenton the salt conditions and DNA sequences. In the presence of magnesiumions, major groove binding facilitates attraction. In contrast, inthe presence of polyvalent cations, minor groove binding serves tocreate charge patterns, leading to condensation. Our findings presenta novel advancement in the field and have broad implications for understandingand controlling nucleic acid complexes  The precise higher-order structuresof nucleic acids are often prerequisites for vital biological processes.A direct example of their organization in biological organisms isthat meter-long DNA helices are compressed and confined inside thecell nucleus in the range of 1016 the tightly bound ion ansatz,17 correlation effects,20 hydration formalism,21 and cation-bridging model,22 have allsucceeded in characterizing and explaining the spontaneous assemblyof DNA molecules. However, each model usually addresses one pertinentaspect of the system and has a limited scope of applicability. Forinstance, the cation-bridging model can only be applied to biogenicpolycations, such as spermine4+, but was not applicableto point-charge cations such as divalent cations. Earlier simulationworks reporting bridging Mg ions23 likelyattributed to the artifacts of the cation parameters. In the KL mechanism,as another example, the binding of cations to dsDNA major groovesassumes a “static” binding that is unrealistic due tothe known cation density fluctuations. As a result, the model failsto predict DNA condensation by minor groove binders or the recentlyreported DNA aggregation in alkaline-earth metal ions .14 In addition, whilethe hydration force proposal24 was basedon experimental observation and theoretical formulation, its causalrelationship with electrostatics is difficult to appraise.25Decades of research have yet to reacha consensus on the physicaloperating principles of the seemingly simple, nonetheless multifaceted,three-component system of DNA, ions, and solvent molecules. Competingtheoretical models, such as the Kornyshev–Leikin (KL) zippermechanism,2+ salts. Perturbing the solvent by addingethanol condenses DNA; raising the cation valence with Co(NH3)63+ or polyamines condenses DNA. To complicateit even more, chemical modifications of the ribose  or the base have been shown to substantially weaken attraction.26 The study of Yoo et al.6 examined theDNA–DNA interactions in various mixed electrolyte solutions.Computing the effects of entropy by performing potential of mean force(PMF) calculations at different temperatures and dissecting the relativecontribution of various molecular species and interactions to thepairwise PMF, they demonstrate that the Na+/spermine4+ mixture of poly-G is governed by electrostatic forces. Inthe presence of spermine4+, they also reported azimuthalangle correlations, concluding that the bridging mechanism leads toDNA condensation in the presence of spermine4+. More recently,we showed that switching to the homopolymeric sequence of poly-A canalso lead to DNA condensation in the presence of divalent ions.14On the other hand, the challenges in arrivingat a unified theorymay be implicated in the diverse ranges of experimental conditionscapable of condensing DNA. Consider the case of random-sequence dsDNAin divalent Mg28 As a result, several distinctive molecularmechanisms have been identified, such as ion bridging with chain-likepolycations,22 the constructive roles of externallybound interfacial cations, and the destructive effects of deep helicalgrooves internalizing cations.30 Albeit powerful, MDsimulations are subject to inaccuracies in the force fields and difficultiesin sampling the multiple time and length scales of the macromolecularsystem.No existing analytical theories can describeall observations.Recent theoretical efforts have focused on using all-atom moleculardynamics (MD) simulations to dissect the different facets of DNA–DNAinteractions.31 in all-atom molecular simulations of DNA–DNA interactions.Our computational approach enabled us to develop a reliable theoreticalprotocol for studying nucleic acid interactions across various conditions.We were able to accurately reproduce DNA condensation/dissolutionexperiments under different conditions and sequences. Expanding uponprevious studies33 that exclusivelyutilized interhelical spacing as the reaction coordinate for conformationalsampling, we included the azimuthal angle as an additional variable,34 giving rise to two-dimensional (2D) free energysurfaces. This added complexity has yielded novel findings that shedlight on the mechanism of DNA condensation. Our simulations indicatethat the azimuthal angle plays a crucial role in positioning the helicesto facilitate attraction. To investigate the mechanism of like-chargeattraction, we compared the recently observed divalent-mediated dsDNAattraction to the well-known polyvalent-induced DNA condensation.We examined the effect of the DNA sequence on the DNA–DNA interactionby contrasting a repeating poly(A)–poly(T) sequence (ATDNA)with a random sequence (MixDNA) that mimics genomic DNA. Similar toref , referred toas ATDNA, and a quasi-random sequence (GCA TCT GGGC TATA AAA GGG andits complement), referred to as MixDNA. We positioned each DNA pairin parallel  and All-atom molecular dynamics simulationswere used to study theinteractions between pairs of double-stranded DNA (dsDNA). Two DNAsequences were investigated: a 20-bp duplex consisting of two homopolymericchains .with ref  : the interhelical distance(d) and the axial rotations of helices relative toone another  to freely move. The fluctuations of the collectivevariables were monitored, and the convergence of the simulations wasassessed by evaluating the amplitudes of the Gaussian heights depositedin the space of the CVs . Detailed descriptions of the WTMD setup are given in the Supporting Information. Details of the WTMD theoryand our application to DNA–DNA interactions can be found elsewhere.35To exhaustively sample theconformational space of DNA pairs, we performed well-tempered metadynamicsimplemented in PLUMED. another 1a,b. For the CVs 1c. WTMD V, we compute the average cation numberdensityr ≡  represents an arbitrarypoint in space and ⟨.⟩ represents the ensemble averageobtained from a 300 ns brute-force MD simulation. We use a spatialresolution of 1 Å.From the particle positions in a simulationbox of volume NA is the Avogadroconstant, and q is the valence of the specified ion.Based on the cylindrical geometry, we compute the average number ofexcess cations condensed on the DNA up to a distance Rc(λ) is the averagecation concentration around a cylindrical coordinate at a radial distanceλ, cbulk is the bulk concentrationof the cation from the center, and L is the lengthof the DNA long axis.Due to the helical nature of the duplex,we transform the density profile to the cylindrical coordinatesq(r). We probe the electrostatic potentialacting at an arbitrary point r1v, forming a shell of 10 Å thickness on the DNA’ssurface. The cutoff is determined based on the Debye length at thesalt condition. From Φ(r), we computed thestored electrostatic potential energy between helices at an arbitraryinterhelical distance, d, asSimilarly, for each point in space, wecomputed the charge densityρ48 where the total entropy is the sum of the following:SMtransvib and SMrovib aretranslational and rotational vibrations of the macromolecule, while SMtopo accounts for the topographical entropy. Due to the well-definedstructure of the double helix, we found the changes in the SMtopo term negligible.To accurately assess the contributionof thermodynamic potentials,we also computed the entropy changes upon assembly. For that, we dividedthe entropic contributions into the conformational entropy of DNApairs and the entropy of the solvent (cations and water). The firstone is estimated using the multiscale cell correlation (MCC) theory,2+ ions and water, we employedthe two-phase thermodynamic (2PT) method.52 In this method, the density ofstate (DoS) is represented by solid-like (Ss(v)) and gas-like (Sg(v)) components, S(v) = Sg(v) + Ss(v), and the entropy is estimatedfrom the DoS associated with the atomic velocities (v) decomposed into contributions from molecular translation (Strans(v)), rotation (Srot(v)), and vibration (Svib(v))51Figure S2). Detailsof the theory can be found in ref ,DNA–DNA interactionscan be modeled as parallel rods. As these interactions are additive,the forces between the long DNA strands increase in strength, resultingin a strong driving force at the macroscopic level, although theyare relatively weak at the base-pair level. Achieving accurate computationalmodeling of these interactions requires capturing a free energy changeof 0.01 to 0.1 kJ/mol/bp, which is challenging due to inaccuraciesin force fields. However, by extensively sampling inter-DNA distancesand orientations using a force field refined based on solution studiesof DNA interactions,14 We compared the ATDNAsequence with a sequence mimicking the genomic DNA, constructed byintroducing an about equal composition of each nucleobase, abbreviatedas MixDNA henceforth. Both sequences are studied under the same saltconditions to provide a fair comparison. The free energy profilesof the two sequences are shown in d ∼ 2.8 nm (14 Thefree energy of binding ΔF = F(2.8 nm) – F(4.0 nm) ≈ (−0.15)kJ/mol/bp agrees well with the estimated value from osmotic stressmeasurements.54 This difference in energybetween the bound and unbound states results in ≈(45–75)kJ/mol of stabilization for DNA pairs of 300–500 bp, whichis sufficient to overcome the thermal energy, leading to condensation,as observed.14Using our approach, we first looked atthe ATDNA, the homopolymeric DNA sequence that shows condensationin experiments.∼ 2.8 nm 2a, consi14While the free energy profile of ATDNA shows attraction,the freeenergy of MixDNA lacks any deep minimum at short DNA–DNA distances2b. The dd, andazimuthal angle, θ. Consistent with the pictures in Figure S3).To provide furtherdetails about the DNA–DNA interactions,we present the free energy landscape in two dimensions 2c,d. TheATDNA duplexes exhibit attraction under the same conditions asMixDNA exhibits repulsion. To investigate the sequence-dependent causalfactors, we analyze and compare the relevant thermodynamic quantitiesof DNA, cations, and water in detail. Given the critical role of thesolvent, we examine the changes in water and cation coordination inaddition to the electrostatic energy stored in the solvation shell3.y-axis) and spans from 0 to 3.5 Å. The second region corresponds tothe bound cation region identified by the radial distribution function(RDF) and spans from 0 to 6 Å (y-axis). The thirdregion is defined based on the Debye length at our salt conditionand spans from 0 to 10 Å. We define the DNA pairs as a condensedstate when the interhelical distance is at 2.8 nm, and the free stateis defined when pairs are at 4.0 nm  between ATDNA and MixDNA . For both sequences, we observed atwo-layer ion distribution around DNA. However, the localization patternsof cations around the two layers are different in the ATDNA and MixDNAduplexes. In the case of ATDNA, a uniform and well-defined distributionof cations is observed in the major groove. On the other hand, inthe case of MixDNA, discrete binding pockets reflecting the heterogeneityof the sequence are evident. The cation distributions of the DNA surfaceshow stronger DNA–Mg interactions in ATDNA , indicating that the ATDNA sequenceprovides a stronger electrostatic field to attract cations. The differencebetween ATDNA and MixDNA is that in the ATDNA, the major groove surfaceis decorated with two highly electronegative sites, forming chargepatterns that do not exist in the case of MixDNA.14 Additionally, ATDNA possesses a wider major groove,55 which results in a shorter phosphate-to-phosphatedistance between the two strands, leading to a higher negative chargedensity at the surface of the DNA. The higher peak intensity in theradial distribution function (RDF)  observed in the case of ATDNA highlights the importanceof the interchain phosphate group distances.To understand thestructural differences between the cation distributionsthat lead to differences in the electrostatic response of the duplexes,we have plotted the ion densities in the form of radial distributionand three-dimensional (3D) number densities  – Sx(4.0 nm) represents the entropy change for each term, with x = total, cation, DNA. The details of computing each entropyterm are explained in the SI. Our findings are reportedin The electrostaticinteraction in the solvation shell favors DNAcondensation in both sequences, but ATDNA provides better enthalpicstabilization on the absolute scale, yet relative energy changes remainsimilar. However, the question regarding how the entropic factorscompare between the two sequences. To assess the entropic factors,we have partitioned the total entropy in the solvation shell (as definedabove) into the components of cation, water, and DNA. We define thechange in entropy as ΔTΔScationMixDNA ≈ 7 kJ/mol makes DNA condensation less likely for MixDNA.ATDNA’s unique topology, with two continuous binding layersand a wider major groove that facilitates dynamic exchanges of condensedcations,34 leads to an overall free energypenalty of TΔScationATDNA ≈2 kJ/mol. As a result, ATDNA offers a more stable condensate comparedto MixDNA under the same salt condition.Based on our analysis, we found that the displacementof watermakes a significant contribution to the entropy and overall stabilityof the condensed phase  a mixture of Mg2+/Na+, denoted as Na–Mg henceforth; (2) a mixture of Mg2+/K+, denoted as K–Mg; and (3) a mixed solutionof Mg2+/Na+/spermine4+, denoted asNa–Mg–Sp. The concentrations of each species in themixture were adjusted to maintain a constant total Cl– concentration in the simulation box. Details of the molecular setupof the mixtures can be found in the Supporting Information. Similar to the pure Mg2+ solution,we used WTMD to construct the FES of the duplex pairs.The surface structure of ATDNA results in a lower electrostatic energyand higher mobility of cations. This property stabilizes DNA pairsin the presence of the pure MgCl+ to condensed ATDNA in Mg2+ leadsto the dissolution of the macroscopic Mg–ATDNA condensates.56 The free energy profile of K–Mg, to someextent, resembles that of Na–Mg, suggesting a similar effecton the DNA–DNA interactions. These observations are reminiscentof MixDNA in pure MgCl2. Similar to MixDNA in MgCl2, no azimuthal correlations are apparent from the FES, asshown in The freeenergy landscape of the ionic mixtures is presented in 4+ is an effective condensingagent that can condense both long and short DNA duplexes, regardlessof whether they are natural or synthetic.57 Our results demonstrate a global minimum at d ∼2.6 nm .Unlike the Na–Mg and K–Mg solutions,the FES of Na–Mg–Spexhibits a distinct minimum at close interhelical distances 4a,b. Thi∼2.6 nm 4f,which∼2.6 nm  In Na–MgTo understand the molecular details underlying the different behaviorsobserved under the various salt conditions, we analyzed the averagecharge densities around the cylindrical coordinate of the H1 duplexin the free state, i.e., when the two duplexes are far apart. We computedthe excess cation charge around the DNA helix from the concentrationprofiles. The density profiles and excess charges under the four saltconditions are shown in 2+ and Na–Mg–Sp, whichshow attraction. The common feature of the attraction cases is thatthe cation charge density is higher, resulting in excess charge accumulationthat effectively screens the negatively charged DNA charges . TheK–Mg mixture mirrors Na–Mg. In the mixture of Na–Mg–Sp,the positive charge is about +71e. These marked differences in overallDNA charge neutralization are consistent with the observed attractiveand repulsive interactions.The analysis of cation charge density reveals acritical differencebetween binary mixtures (Na–Mg and K–Mg), which showrepulsion, and pure Mg2+ . We observe subtle differencesin ion atmospheres between K+ and Na+. Specifically,K+ tends to be situated around the minor groove (S7), exhibiting a more localized distributioncompared to Na+, which shows diffusive binding in the proximityof the phosphate backbone .The addition of Nar groove 7e vs. S7 Figure S vs. 6.2+ ionskeep a fair distance away from the DNA surface, e.g., peaking at an∼4.2 Å distance compared with an ∼2.4 Å surfacedistance for Na+ and an ∼2.8 Å surface distancefor K+ (see S7). This can beattributed to the strong hydration shell of Mg2+, whichremains largely intact when bound to DNA. Unlike Mg2+,the Na+ ions have a weaker solvation shell, leading tofrequent dehydration events and almost equal Coulombic forces betweenhexahydrated divalent magnesium cations and dehydrated monovalentsodium cations. The larger size of K+ ions leads to a weakerhydration shell compared to Na+.58 This property also facilitates the direct binding of K+ ions to the DNA surface, causing them to compete with Mg2+ ions for DNA binding .How a monovalent ion competes with a divalention remains an interestingquestion. A careful look at the SDFs shows that Mg4+ to the mixture helpsto partially regain most of the lost territories of the magnesiumbinding sites in the major groove. This observation is better seenwhen we look at the SDF . The interdigitated charge patterns create “zipper-like”charge–charge correlations via orientational coupling. DNAassembly is driven by electrostatic attraction between correlatedcharges of opposite signs. It is worth noting that in spite of resemblingpositional arrangement, our proposed mechanism is physically differentfrom the KL zipper model. First, the KL model starts with cationsalready bound in the major groove based on empirical observations,while the major groove cation binding here is substantiated by physicalMD simulations. We further show cation binding to be sequence- andcation-dependent, which is a fundamental difference between ATDNAand MixDNA. Second, the KL model presumes static cation binding inevenly spaced sites that are unphysical, while the cations in themajor groove here are observed to retain high levels of mobility,evident from their continuous spatial distributions and entropy calculations.Our studies indicate that the cation dynamics not only make key entropiccontributions but also allow exchanges (hops) between adjacent helices.Cation dynamics is thus a novel observation of this study, which requirescertain surface features and shallow cation binding.Specifically, homopolymeric ATDNA lines up the partialchargesin the major groove in a continuous and periodic fashion, which notonly enhances cation binding but confers significant mobility to hydratedMgdefacto multifaceted, and perhaps more remarkably, nucleicacids prove to be capable of modulating molecular interactions viaa variety of surface restructuring strategies. For example, drasticchanges can be made by forming multistranded structures such as triplexand quadruples, which have been shown to condense in divalent andeven monovalent counterions.60 Biochemical modificationssuch as methylation and hydroxylation occur widely in biology andhave been shown to promote interhelical repulsion. Our study hereaddresses a more subtle perturbation of the structure via the DNAsequence alone, which nonetheless demonstrates the ability to turnrepulsion into attraction in divalent salts. Such multistranded structures,modifications, and repeating sequences are abundant in biology. Itis important to recognize the peculiarities of these noncanonicalstructures to elucidate their functional mechanisms at the molecularlevel. Furthermore, the versatility of nucleic acids in modulatinginteractions is expected to be able to assist with the synthesis offunctional or therapeutic assemblies, such as DNA origami and lipidnanoparticles.On thewhole, while often deemed to be dominated by the electrostaticsof the phosphate backbone, nucleic acid interactions are


The microenvironment of duodenum was assessed by measuring oxygen saturation using T-Stat, serum inflammatory markers and zonulin for gut permeability. We observed that bacterial overload was correlated with increased serum zonulin (p = 0.061) and higher TNF-α (p = 0.054). Moreover, reduced oxygen saturation (p = 0.021) and a systemic proinflammatory state [increased total leukocyte count (p = 0.031) and reduced IL-10 (p = 0.015)] characterized the duodenum of hyperglycemic. Unlike stool flora, the variability in duodenal bacterial profile was associated with glycemic status and was predicted by bioinformatic analysis to adversely affect nutrient metabolism. Our findings offer new understanding of the compositional changes in the small intestine bacteria by identifying duodenal dysbiosis and altered local metabolism as potentially early events in hyperglycemia.The gut microbiome influences the pathogenesis and course of metabolic disorders such as diabetes. While it is likely that duodenal mucosa associated microbiota contributes to the genesis and progression of increased blood sugar, including the pre-diabetic stage, it is much less studied than stool. We investigated paired stool and duodenal microbiota in subjects with hyperglycemia (HbA1c ≥ 5.7% and fasting plasma glucose > 100 mg/dl) compared to normoglycemic. We found patients with hyperglycemia (n = 33) had higher duodenal bacterial count ( The disease is preceded by the state of intermediate hyperglycemia known as early diabetes or pre-diabetes. This often goes undiagnosed but presents an opportunity to understand the early effects of disease on biological health of humans. Even though the precise mechanism of disease is not well understood, evidence from experimental and clinical studies show that gut microbiota importantly determine the pathogenesis and course of metabolic diseases including diabetes2. Alteration of gut microbiome could induce insulin resistance (IR) in mice, implying an important role of microbiome in the development of diabetes. Similarly, the transplantation of stool microbiota from lean donors to patients with Metabolic syndrome augmented gut bacterial diversity and improved insulin sensitivity (IS)3. Such microbiota-attributed impact is at least in part due to altered microbial metabolites, promoting or ameliorating IS. For example, decrease in IS is correlated with decreased abundance of butyrate producing bacteria in the gut4.Type 2 diabetes mellitus (T2DM) is a chronic progressive metabolic disorder characterized by the body’s impaired mechanism of regulating sugar levels. It is rapidly becoming a major health care burden both in developing as well as in developed economies5. It has been shown that impaired gut barrier integrity has a microbial-origin, resulting from a triggered inflammatory response via lipopolysaccharide6, which compromises the functionality of tight-junction proteins and causes IR. Besides physical barriers, tissue oxygenation is a less commonly studied factor, such that hypoxia promotes inflammation and insulin resistance.In fact, host-commensal-pathogen interactions in the gut are also determinants of increased gut permeability, which is an underlying feature of chronic metabolic diseases7. There have only been few studies focused on upper gut, since techniques such as endoscopic duodenal mucosal resurfacing (DMR) and placement of duodenal sleeve, both of which bypass a possibly ‘diseased duodenum’, have shown beneficial improvement in glycemic control9 and restoration of metabolic homeostasis. While existing evidence around the role of duodenum in diabetes is focused on local nutrient effects via the gut hormonal axis10, indirect effects through dysbiosis and immune stimulation are plausible. Recent findings of metformin’s ability to induce glucoregulatory effect via modifying small intestinal bacteria11, and mechanism of action of resveratrol acting via duodenal specific Sirt1, enhancing insulin sensitivity12 make such research of utmost significance.For a long time, microbiome research has heavily relied on stool samples for studying gut bacterial profile, however stool microbial communities reflect the microbiota resembling the distal gut and do not capture the mucosa associated microbiota (MAM) of the upper gut. Previous investigations have indicated MAM communities in upper gut as risk factors for impaired glucose metabolism13. Associations starting from pre-diabetes are less likely to suffer from a cause-effect bias where supra-physiological glucose levels of intestinal fluids induce changes in microbiome.The links between hyperglycemia and the factors discussed above are likely to be bidirectional, such that the gut physiology impacts glycemia and glycemic states alter gut physiology. Such relationships, related to MAM, hypoxia, or epithelial permeability, are best explored in the upper gut where the surface area is large, nutrient signaling is coupled to gut-hormonal and gut-neural axes, and host-microbe barriers are not strong as in the colon14, in cigarette smokers15, small intestine bacterial overgrowth16, Celiac Disease17, pancreatic cancer18, and enteropathy20. This growing body of evidence highlights that upper gut microbial alterations could be causal in disturbing the immune and metabolic signaling contributing to diseases. Our characterization of the overall microbiome in proximal duodenum and paired stool samples in hyperglycemia adds to the lacking body of knowledge about the role of duodenum and the associated microenvironment in early disease state. It is the first such study in Indians. To address the specific question of whether duodenal microbial communities and microenvironment  are different between people with and without hyperglycemia, we recruited subjects who underwent Upper GI endoscopy as part of their standard screening for GI symptoms from PGIMER to obtain duodenum biopsy and paired stool samples for 16S rRNA gene microbiome analysis. Overall, we identified dysbiotic duodenal microbial signatures and provided new insights into the associations of altered microenvironment and microbiota in hyperglycemia.In this study, we investigated the hypothesis that duodenal bacterial dysbiosis and altered microenvironment may be associated with hyperglycemia of pre-diabetes and diabetes (HbA1c ≥ 5.7% and fasting plasma glucose > 100 mg/dl). Prior research examined duodenal bacteria primarily in functional dyspepsiaA total of 116 samples were sequenced, of which 108 were paired stool and biopsy samples from 54 subjects and remaining 8 were negative controls. Negative controls are samples that contain all DNA extraction required reagents but have no biological material. Paired biopsy and stool samples from each subject were analyzed for 16S microbial analysis. There were 33 hyperglycemic and 21 normoglycemic and none of the subjects had taken any antibiotics in the last 6–8 weeks prior to endoscopy. The endoscopic features of duodenum were found normal in both the study groups. None of the subjects showed severe inflammation or any duodenal related pathology under the histopathological examination. The microbial characterization of a total 108 paired stool and duodenal samples using targeted amplification of V3-V4 of 16SrRNA gene generated 9,926,256 sequences which were assigned to 795 Amplicon Sequence Variants (ASVs).The analysis of 16S amplicon data was carried out to study the bacterial composition of duodenum microbiome in both hyperglycemic and normoglycemic subjects. The relative abundance of dominant phyla was represented by Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria, and Fusobacteria in both the study groups. There were also minor phyla which included Lentisphaerae, Tenericutes, Epsilonbacteraeota, Chlamydiae, Verrucomicrobia, Patescibacteria, constituting < 1% and < 5% of microbiota in hyperglycemic and normoglycemic subjects respectively . The PCoA data segregated along Axis 1 and explained 38.1% of the variation in sample clustering  while Firmicutes was significantly decreased  in duodenum. The relative percentage of Fusobacteria and Actinobacteria accounted for 8% of MAM and the former remained undetected in stool  while the latter  only constituted 1.6% of the stool profile. On the other hand, Bacteroidetes constituted approximately 80% of the stool microbial data with Firmicutes and the proportion of Bacteroidetes was one-third of the stool microbiota  in duodenum  and found two distinct clusters representing stool and duodenum  and 153 taxa were found to be differentially abundant in the two sample types   and core  bacterial composition of the duodenal samples between hyperglycemic and normoglycemic groups. In contrast, the microbial profile of stool samples was not different between the study groups . To better interpret these findings, we also calculated the within-group dispersion (p = 0.172) and found it had no bearing on PERMANOVA results. Thus, bacterial differences in the duodenal samples of hyperglycemic and normoglycemic groups were significantly greater than differences observed within groups  Fig. a. Next, p = 1.7e−06)  and higher duodenal diversity (p = 0.099) in hyperglycemics compared to normoglycemics, however this was not found to be statistically significant  or in diversity (p = 0.173) was observed in the stool microbiome between the two groups , which is an important determinant of altered gut profile. We observed F/B ratio did not differ between the two study groups  Fig. c. We alsant Fig. d. On the p = 0.17 was obse3 bacteria/ng DNA) in the duodenum of hyperglycemics than normoglycemic  subjects , higher FPG  and higher PPG  was detected in subjects with high bacterial load  in hyperglycemics. This microbial alteration was exacerbated by the enrichment of pathogenic species such as a 30-fold increase in Acinetobacter haemolyticus  and Escherichia/Shigella_NA  as well as 18-fold rise in Dolosigranulum pigrum , and nearly fourfold spike in Streptococcus cristatus  in the duodenum of hyperglycemic subjects. At family level, there were 6 families that were enriched in normoglycemic, namely, Coriobacteriaceae , Akkermansiaceae , Nocardiaceae , Tannerellaceae , Caulobacteraceae  and Pseudonocardiaceae . Furthermore, orders such as Pseudomonadales , Pseudonocardiales  and Caulobacterales  were elevated in normoglycemic. No differences were found at phylum or class level in the duodenal microbiome of hyperglycemic and normoglycemic subjects . Notably, there was 23-fold reduction in the abundance of beneficial bacteria like p = 0.031). In addition, the levels of the inflammation resolution cytokine, Interleukin 10 (IL-10) were found to be significantly reduced (p = 0.015)  in hyperglycemics, which showed a trend of positive correlation with HbA1c    than normoglycemic subjects (15) Fig. a. There 66) Fig. b. Additip = 0.021) compared to normoglycemic   Fig. e.22. We observed subjects with normoglycemia and those with hyperglycemia did not differ in terms of zonulin levels   and zonulin correlations also extended to obesity associated parameters such as body mass index , waist circumference , and hip circumference  , FPG , and PPG . Similarly, another important pathway, involved in the synthesis of aromatic amino acids exhibited an unfavourable relationship with HbA1c , FPG , and PPG . Besides these negative associations, a favourable link of purine metabolism with HbA1c , FPG , and PPG  was found by association analysis, which also extended to pathway related to translation, HbA1c , FPG , and PPG   producer35, that contributes to small intestinal motility36, which is an important determinant of blood glucose37.This is further substantiated by the significant enrichment of genera that were associated with gut and metabolic improvement in normoglycemic, such as 38. Families Prevotellaceae, Succinivibrionceae, Lachospiraceae, Ruminococcaceae dominate in both normoglycemic and hyperglycemic. At class level, Bacteroidia, and Clostridia were prominent and to a lesser extent, Gammaproteobacteria, Actinobacteria and Negativicutes classes were also present. The stool microbial community was similar between the groups while the duodenal profile showed important microbial differences associated with glycemia. This could be caused by the duodenum and pancreas shared embryological, functional, vascular and structural relationship, which could affect the entero-insular signaling, crucial for maintaining glucose homeostasis39.The stool profile was consistent with the already available data of an Indian gut40. Moreover, the F/B ratio, which is one of the hallmarks of the dysbiotic gut profile41 remained unchanged between hyperglycemic and normoglycemic but was found to be higher in duodenum, indicating that disease specific cues were detected at a higher signal strength in the duodenum than in stool.We found greater inter-individual variability in the mucosal microbial composition similar to the findings of Vaga et al.42. Excess of bacteria may be related to immune activation that may have an etiological role in diabetes43. The apparent high duodenal bacterial load observed in hyperglycemia and the correlation between bacterial burden and inflammatory marker  supports the above observation. Further, the positive correlation between TNF-α and HbA1c corroborated such an association. We also noted reduced anti-inflammatory IL-10 levels, which is shown to increase insulin sensitivity and affect peripheral glucose metabolism45. Together, our results suggested the duodenal microenvironment to be inflammatory, overpopulated with bacteria, and with reduced anti-inflammatory signals in hyperglycemic condition.To better understand the full potential of upper gut microbes to modulate human health, the factors influencing the interactions between mucosal microbes and their environment are worth exploring. Indeed, the prominent one is the amount of duodenal mucosal bacteria due to the aggregates of lymphoid follicles residing underneath46, described the zonulin pathway as a mechanism of innate defense, flushing out the microbes with water using hydrostatic pressure gradients. In our data, we did not find any difference in zonulin levels between the groups, as observed in previous reports47 or any association with taxa. Interestingly, higher zonulin levels showed a trend of association with increased mucosal bacterial load. The relatively small size of the dataset for zonulin measurements is likely to underestimate the relationship between permeability and microbial markers. To our observation, our data was at least sufficient to provide a robust validation to the previously addressed associations between zonulin and anthropometric measurements related to obesity48.Another important microenvironment parameter influenced by intestinal bacteria is gut permeability. In this study, we measured serum zonulin, a marker of small intestinal permeability. Research by Asmar et al.49. On the other hand, microbes also actively participate in shaping the mucosal environment by degrading mucus or triggering AMP production50. Our observations showed a lower duodenal tissue oxygenation in hyperglycemia. This decrease could be due to the reduced perfusion of the gut or due to increased local utilization of oxygen51, which could not be distinguished by the data. Thus, the question of whether decreased oxygenation is a cause, or consequence, of the altered microbial profile or metabolism in the duodenum, remains to be explored. It is interesting that the oxygen saturation values for both groups are lower than those reported in Western studies52. Since this is the first such study from South Asia, it is conceivable that bacterial overload and lower oxygenation may be frequent in low-middle income tropical countries. Future multi-centric studies should explore this further.Besides physical barrier, tissue oxygenation maintains the functional balance of mucosa and directly shapes microbial community as exemplified by the increased amount of dissolved oxygen after RYGB surgery that favors the proliferation of Proteobacteria phyla53. To our observation, we found diminished ability of synthesizing aromatic amino acids  in hyperglycemia which could impact host physiology. Small molecules with potent biological effects are derived from these aromatic amino acids. For example, tryptophan derived metabolites  are important modulators of epithelial integrity and mucosal immune response54. Another key pathway implicated in altered β-cell metabolism is the TCA cycle pathway55, which was found to be reduced in the duodenum of hyperglycemic. Such abberant change in local duodenal metabolism under high glycemic condition could be brought on by altered microbial metabolism. The changed metabolic state could possibly also disrupt the core imbued duodenal pathways of nutrition sensing and entero-insular signalling, critical for maintaining glucose homeostasis56.There is not a considerable body of clinical data to link the pathogenesis of metabolic diseases to microbial disturbances in the upper gut or the associated environment. In this study, we have investigated this relationship and predicted the metabolic state of the duodenum in hyperglycemic condition. In duodenum, the main source of energy is amino acids such that 30 to 50 percent of amino acids do not enter portal circulation57. Secondly, the study was performed in a limited number of subjects and directionality of associations could not be concluded from the data. Third, zonulin levels represent the small intestinal permeability and are not specific only to duodenum but in the jejunum as well, thus it cannot be directly linked to duodenal microbiome. Fourth, the study is cross-sectional in nature and captures the intestinal bacterial composition at a single time point.We acknowledge some important limitations of the study. First, the age of the normoglycemic group was significantly lower than that of the hyperglycemics. This could be attributed to the increased prevalence of hyperglycemia with aging. To account for effect of age, we have adjusted the age variable while analyzing data using DESeq2, as previously reported in small bowel microbiome studyThere are multiple factors which define strengths of the study. First, we included microenvironment variables  alongside the microbiome, unlike most prior duodenal microbiome studies. Second, the use of negative controls during DNA extraction provided an additional confidence to the results of the study, as low biomass biopsy samples are prone to contamination. Third, we analyzed hyperglycemia without remaining restricted only to diabetics, to study the early disease associated microbial changes which could be leading to diabetes and most importantly, the paired assessment of duodenal and stool bacterial profile was performed for the enrolled patients.In conclusion, variability in the duodenal mucosa associated microbiota profile showed association with glycemia. The assessment of small intestinal mucosa associated microbiota, with the inclusion of a quantitative approach, merits deeper investigation as a health determinant. Understanding the compositional changes in the small intestine bacterial population is an important area of research, especially in the context of human nutrition and to find future link to human health and disease.In this cross-sectional study, we recruited 69 subjects after obtaining informed consent in 2018–19 according to the guidelines of the Declaration of Helsinki, 1975. The study was conducted in compliance with the Indian Council for Medical Research guidelines, Good Practices for Clinical Research in India. The study was approved by the Ethics Committee of PGIMER . Written informed consent was obtained from all study participants.58. Overall, 33 established hyperglycemic and 21 normoglycemic were included in the study. As shown in Table The inclusion criteria included adult subjects (18–64 years) who underwent Upper GI endoscopy as part of their standard screening for GI symptoms, anemia as well as to rule out celiac disease or GI bleeding. Subjects who were pregnant, lactating, had celiac disease, colon cancer and gastrointestinal (GI) bleeding were excluded. After eliminating subjects with a history of consumption of antibiotics in the last 6–8 weeks, absence of paired duodenum/stool sample and low read sequencing depth (< 2000), 54 subjects with paired samples were finally analyzed. Based on the blood parameters; fasting plasma glucose , postprandial glucose  and glycated hemoglobin , subjects with FPG > 100 mg/dl and HbA1c ≥ 5.7%; were classified as hyperglycemic, while those with FPG ≤ 100 mg/dl and HbA1c < 5.7%; were classified as normoglycemic59. Negative controls were maintained in each batch of DNA extraction. For library preparation, V3-V4 region of 16S rRNA was selected and paired-end sequencing was performed as per the Illumina approved protocol for 16 s rRNA marker gene survey60 on MiSeq platform . The final library was reconstituted using the Tris buffer at 4 nM. The loading concentration of the library was 8 pM with 15–20% PhiX.Duodenal biopsies from the D2 region were obtained endoscopically using the Olympus endoscope GIF-HQ190. The biopsy was collected in RNAlater for microbial analysis and for histology in 10% formalin. The stool sample was collected in a polypropylene container and stored at − 80 °C without preservatives. Blood samples were collected in additive free red capped vacutainers to obtain sera. The extraction of DNA from stool and biopsy samples was performed using Qiagen Stool Mini kit and DNeasy Blood and Tissue kit respectively, with the additional step of bead-beating61, raw sequences were filtered with following parameters; maxEE = c, trunclen = c, trimleft = c, maxN = 0, truncQ = 2, rm.phix = T and the settings for remaining steps were default. The amplicon sequence variant (ASV) tables from each of the two duodenum and stool runs were then combined and taxonomy was assigned using SILVA database (Release 132). Subsequently, the phyloseq object containing the phylogenetic tree, constructed using the Neighbor joining method, was decontaminated using the "decontam" package after chimera removal. In decontam, there are two approaches for decontamination; i.e., frequency and prevalence-based. The combined approach was applied for stool, and the recommended prevalence-based method was used for low-biomass duodenum samples. Taxa that appeared more than three times across samples and had a prevalence of more than 5% were included for total microbiome analysis, and taxa that had a prevalence of at least 50% was used for core microbial analysis.Using DADA2 pipeline62. The inter-sample Bray Curtis measurements were computed using the microbiome package. The relative abundance at phylum level was used to calculate Firmicutes/Bacteroidetes ratio in stool and biopsy samples.R was used for statistical analysis and ggplot2, ggpubr packages were used for data visualization. Based on the distribution, clinical parameters were expressed as medians with interquartile range [Q1-Q3] or mean (standard deviation). Histogram was constructed to represent the top 10 taxa with the highest proportion at different taxonomic levels in duodenum and stool samples. At a rarefied depth of 13,188 ASVs/sample, alpha diversity measures (Observed Species and Shannon Diversity) were calculated. To compare paired quantitative samples, non-parametric Wilcoxon Sign Rank test was applied. For beta diversity, the rarefied object was hellinger transformed and Principal Coordinate Analysis (PCoA) was used to discover grouping patterns. Variability in the community structure was assessed by permutational analysis of variance (PERMANOVA) using adonis function in vegan packageUsing the DESeq2 package, the differential abundance analysis on untransformed species level phyloseq object was performed between stool and biopsy samples. The differential abundance analysis was also performed between hyperglycemic and normoglycemic to find characteristic features at different taxonomic levels with respect to glycemia in stool and duodenum samples. Age and sequencing batch effects were adjusted wherever required and p < 0.05 was regarded statistically significant.63 were used with 15 ng of template in a Roche LightCycler 480 to set up a reaction. The amplification conditions were 3 min at 95 °C followed by 40 cycles of 95 °C for 15 s and data collection at 60 °C for 20 s. Using a previously recommended cut-off of normalized 103 bacteria for determination of small intestinal bacterial overgrowth64, the bacterial count data of hyperglycemic and normoglycemic was categorized as ‘high bacterial load’ and ‘low bacterial load’ and compared using Chi-square test with Yates correction. Further, the levels of HbA1c, FPG and PPG were compared between the 'high bacterial load’ and ‘low bacterial load’ groups.A series of tenfold dilutions of recombinant plasmid (full length 16S rRNA gene of E. coli cloned in pRS315 vector) were used to create a standard curve. The universal 16S primers, eubF (GTGSTGCAYGGYTGTCGTCA) and eubR (ACGTCRTCCMCACCTTCCTC)The Luminex assay was performed using MILLIPLEX MAP Human Cytokine/Chemokine HCYTOMAG-60 K-8 Panel to measure the concentrations of Interferon-γ, Interleukin-1β, Tumor Necrosis Factor-α, Interleukin-8, Interleukin-4, Interleukin-5, and Interleukin-10. The software xPONENT 3.1 (Luminex corp.) was used to examine the data and the levels of inflammatory markers were compared between the two study groups. The relationship between these factors and anthropometric measurements, glycemic markers, and bacterial load was examined by Spearman correlation test.65. Only pre-endoscopic sera samples were used to measure zonulin (16 hyperglycemic and 12 normoglycemic). The spearman correlation test was used to test association between zonulin and anthropometric measurements and bacterial load.The FDA-approved T-Stat (Spectros), was used to measure duodenal mucosal oxygen saturation (D2). Only 47 patient’s mucosal oxygenation could be measured due to technical difficulties . After suppressing peristalsis with a single dosage of hyoscine butylbromide (Buscopan- 10 mg/ml), multiple measurements were taken and an average of three readings per individual was used for analysis. The gut permeability assay using serum zonulin was performed using ELISA kit https://www.genome.jp/kegg/ko.html). The samples were normalized by overall functional abundance (to compare the relative abundance of predicted pathways between samples) and the anticipated functional profile was adjusted for 16S copy number variability. A linear regression model adjusted for sequencing batch effects and fluctuation in the fraction of unused taxonomic units (no hit in the reference database), was used to find pathways that were linked to glycemic status. Subset of data was plotted as correlation graphs using psych package and the entire data was represented as heatmap using the pheatmap package.Based on the 16S sequencing data, Tax4Fun (t4f.) in "themetagenomics" package was used to predict metabolic capabilities of duodenal bacteria. We mapped the taxonomic abundance and annotated the functional content using KEGG orthologues (Supplementary Information.


Moreover, rES reduced genetic diagnostic costs by 1.5% (€85 per neonate).The introduction of rapid exome sequencing (rES) for critically ill neonates admitted to the neonatal intensive care unit has made it possible to impact clinical decision-making. Unbiased prospective studies to quantify the impact of rES over routine genetic testing are, however, scarce. We performed a clinical utility study to compare rES to conventional genetic diagnostic workup for critically ill neonates with suspected genetic disorders. In a multicenter prospective parallel cohort study involving five Dutch NICUs, we performed rES in parallel to routine genetic testing for 60 neonates with a suspected genetic disorder and monitored diagnostic yield and the time to diagnosis. To assess the economic impact of rES, healthcare resource use was collected for all neonates. rES detected more conclusive genetic diagnoses than routine genetic testing , in a significantly shorter time to diagnosis (15 days (95% CI 10–20) vs. 59 days (95% CI 23–98, Conclusion: Our findings demonstrate the clinical utility of rES for critically ill neonates based on increased diagnostic yield, shorter time to diagnosis, and net healthcare savings. Our observations warrant the widespread implementation of rES as first-tier genetic test in critically ill neonates with disorders of suspected genetic origin.The online version contains supplementary material available at 10.1007/s00431-023-04909-1. Genetic disorders are frequent causes of neonatal morbidity and mortality, and disease presentations are often undifferentiated at birth . Since gApproximately 2.5% of newborns is admitted to neonatal intensive care units (NICU) in the Netherlands . The preBesides clinical implications, implementation of improved diagnostic tests can also have health economic impact. Gonzaludo et al.  have shoBased on several studies showing that rES provides a faster diagnosis, enabling timely precision medicine aiming to decrease morbidity and mortality of infants with genetic disorders , 11–13, We performed a multicenter prospective parallel cohort study, in which we assessed the clinical utility of rES compared to conventional genetic testing, i.e., routine genetic testing/the genetic trajectory based on decisions of the clinicians. In order to compare these two genetic trajectories, all study participants received both conventional genetic testing and rES in parallel Fig. . This stThis study was approved by the Medical Research Ethics Committee Arnhem/Nijmegen under file number 2016–2486/ NL57511.091.16.We studied 60 neonates admitted to a NICU in five out of ten centers in the Netherlands between May 2017 and January 2019. This sample size was calculated based on a two-sided chi-square test, using a power of 80% and a significance level of 0.05. The diagnostic yield of routine genetic testing was estimated to be 5% and 25% for rES. The sample size calculating resulted in a minimum of 55 patients, with a significance level of < 0.001. Taking into account a possible drop out of 10%, during the course of the study, 60 patients were recruited. Criteria for inclusion were a postnatal age less than 3 months at presentation and high suspicion of a genetic disorder . In addition, EDTA blood samples of both biological parents were required for participation. Exclusion criteria were a previously  established genetic diagnosis or a clinically phenotype highly associated with trisomy 13, 18, 21, or monosomy X. Parents were informed about the study by the attending clinician, in consultation with a clinical geneticist, if routine genetic testing was indicated. Written informed consent was obtained for all participating families.Clinical features were scored using the human phenotype ontology (HPO) terms  Supplem. CA wereThe rES procedure was performed in the ISO15189 accredited genetic diagnostic laboratories affiliated to the five NICUs. Whereas minor technical differences exist between centers, such as, for instance, different enrichment kits or sequencing equipment used, the overall procedures were similar –19. ImpoFor each neonate, we monitored the routine genetic diagnostic trajectory . In contrast, the TTD was only determined for neonates who received a conclusive diagnosis. The latter was chosen as this reflects an objective end point of genetic diagnostic care for index patients. The TTD was measured from the moment the first genetic test was requested by the involved clinician until the return of the final conclusive genetic diagnostic report. Of note, in case ES was request in the conventional diagnostic strategy, only the rES was performed, and the TAT of routine ES was censored to overcome the need to perform the ES procedure twice. To determine this censored TAT of ES in conventional diagnostic testing, the TAT of ES was determined from a random, anonymized, set of individuals, unrelated to this project, but equal in size of the number of neonates receiving rES. This resulted in a TAT for ES of 105 days (95% CI 96–113 days).Healthcare consumption was collected from the electronic patient file of 56/60 patients. For four neonates, these data could not be collected, and therefore, these patients were excluded from the cost analyses. Healthcare activities were linked to their unit prices (index year 2020) retrieved from the Dutch Healthcare Authority (Nederlandse Zorgautoriteit (NZA)) and the National Healthcare Institute (Zorginstituut Nederland (ZiN)) , 22, aftFor all healthcare data, average costs per neonate were calculated, including the minimum, maximum, and median. Costs were divided into seven categories based on their type of healthcare activity: diagnostics, hospitalization, consult, surgery, medicines, genetic, and other healthcare costs. Per category, a percentage of total healthcare costs was calculated. For the category “genetic costs,” the types of genetic tests performed were also retrieved to provide more detailed insight to the build-up of costs associated with genetic testing.Next, two cost analyses were performed, firstly the economic impact of implementation of rES compared to the conventional genetic testing, and secondly, the evaluation of the timing of expenditure for genetic testing, differentiating between costs made during the neonatal period (first 28 days starting at birth) and costs during the post-neonatal period with a maximum follow-up period of 2 years after birth.t test was performed to analyze whether the difference in TAT and costs between the two diagnostic trajectories were significant.Data analysis of the clinical variables was performed in Excel (version 2016), and the cost analysis was performed in R (version 4.0.3) . In morep < 0.001), as expected given our study purpose and the relation between genetic disorders and CA [A total of 60 newborns admitted to the NICU with clinical features suggestive for a possible genetic disorder were enrolled in this study. Demographic information is presented in Table s and CA . PatientFor all 60 neonates, the genetic diagnostic trajectory started in the neonatal period, at an average age of 8 days (95% CI 6–10 days). From this start point onwards, the neonates received both routine genetic testing and rES in parallel, allowing to directly compare outcome measures as neonates served as their own controls.In routine genetic testing, a total of 112 genetic tests were performed, resulting in an average of 1.87 tests per patient , providing higher diagnostic yield than the routine genetic tests  reduction in genetic costs by 1.5% . We subsequently aimed on gaining insight into the timing of healthcare utilization and associated costs  were substituted by the costs of trio-based rES Supplementary file2 (XLSX 19 KB)Supplementary file3 (XLSX 16 KB)Supplementary file4 (XLSX 16 KB)Below is the link to the electronic supplementary material.


P = 0.008) and body mass index (BMI)  were independently associated with MAFLD. Furthermore, only the alcohol use disorders identification test (AUDIT) was able to identify ALD . Our study revealed that health checkups, including measurement of ALT, BMI, and AUDIT, are important for screening MAFLD and ALD in younger generations.We aimed to assess metabolic dysfunction-associated fatty liver disease (MAFLD) and alcohol-related liver disease (ALD) prevalence in young male adults and the role of health checkups in disease screening. We recruited 313 male graduate students at Gifu University in April 2022. With hepatic steatosis diagnosed by ultrasonography, MAFLD and nonalcoholic fatty liver disease (NAFLD) were diagnosed based on health checkup data, and ALD was diagnosed with alcohol consumption > 30 g/day. The ability of each variable to identify MAFLD, NAFLD, and ALD was assessed using logistic regression and receiver-operating characteristic curve analyses. Participants’ mean age was 23 (± 4) years, and MAFLD, NAFLD, and ALD prevalence was 11%, 17%, and 1%, respectively. Among Japanese male young adults, alanine aminotransferase (ALT) (odds ratio [OR] 1.04; 95% confidence interval [CI] 1.01–1.07;  Although CLD remains asymptomatic for a long period, when it eventually progresses to cirrhosis, patients experience painful clinical conditions, such as malnutrition, sarcopenia, varices, ascites, and hepatic encephalopathy, which result in impaired quality of life, frequent hospital admission, and poor prognosis3. The etiology of CLD is dramatically shifting from hepatitis B/hepatitis C viral infections to alcohol-related liver disease (ALD) or nonalcoholic fatty liver disease (NAFLD) due to the implementation of hepatitis B vaccination, advances in hepatitis C treatment, increasing obesity and metabolic syndrome incidence, and rising alcohol misuse1. The Global Burden of Disease data analysis demonstrated that the cause of CLD among 15–29-years-olds has also shifted worldwide from hepatitis B infections to NAFLD4. Therefore, early detection and intervention of NAFLD and ALD is an unmet clinical need, especially in young adults, for reducing CLD burden.Chronic liver disease (CLD), characterized by chronic liver inflammation, affects 1.5 billion people globally, accounting for nearly 2 million deaths due to liver cancer and cirrhosis6. Therefore, MAFLD is diagnosed with hepatic steatosis and the presence of at least one risk factor, such as obesity, type 2 diabetes mellitus, and metabolic dysregulation6. Recent evidence showed that the MAFLD criteria identified patients with more advanced liver fibrosis and worse prognosis than the NAFLD criteria only8. Since the diagnosis of MAFLD does not rely on alcohol consumption, recent studies emphasize that alcohol consumption is an independent factor for stratifying the risk of liver fibrosis and mortality in CLD9. Although adverse effects of MAFLD on CLD have been demonstrated8, very few attempts have been made on screening and assessing the prevalence of MAFLD among young adults. Additionally, although MAFLD was associated with the worsening symptoms of CLD, guidelines or best practices for the relatively new criteria have not been established.With the accumulated evidence of NAFLD, a panel of international experts from 22 countries proposed “metabolic dysfunction-associated fatty liver disease” (MAFLD) as a new term to identify patients with fatty liver disease at risk for severe outcomesIn Japan, a mandatory annual health checkup for all populations has been established and provided even for young adults, based on the Health and Safety Act and the Occupational Health Act. The primary aim of this study was to determine the prevalence of MAFLD and ALD in young Japanese male adults. The secondary aim was to establish robust screening methods for MAFLD and ALD during the annual health checkups.This cross-sectional study included male graduate students who had entered Gifu University  and underwent an annual health checkup for freshmen in a graduate program in April 2022. The following inclusion criteria were used: an incoming student at Gifu University Graduate Program of 2022, aged 18 years or older, self-identification as Japanese, and completion of all examination items including blood sampling and health questionnaire that included present medical treatment. All male students attended the health checkup underwent abdominal ultrasonography  administered by a hepatologist completely blinded to the results of this study, along with a questionnaire on alcohol consumption. Biochemical assessments included aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol, hemoglobin A1c (HbA1c), and fasting or random glucose levels.Of the 613 incoming students at Gifu University Graduate Program of 2022, 120 had no annual health checkup because these students were part-time or executive students who have a health checkup at their workplace or international student who could not attend the scheduled health checkup. In addition, 106 females were initially excluded from the study and 60 students were excluded based on the inclusion and exclusion criteria. Finally, 313 Japanese male graduate students were included in the analysis  ≥ 23 kg/m2 according to the Asian cutoff value. Type 2 diabetes mellitus was defined as HbA1c ≥ 6.5% or use of a specific anti-diabetic treatment. Metabolic dysregulation was defined as the presence of at least two of the following criteria: waist circumference ≥ 90 cm, blood pressure ≥ 130/85 mmHg, TG level ≥ 150 mg/dl, HDL-C level < 40 mg/dl, prediabetes with fasting blood glucose from 100 to 125 mg/dL, random blood glucose 140–199 mg/dL, HbA1c from 5.7 to 6.4%, or any specific treatment related to these conditions5.The diagnosis of MAFLD was based on the diagnostic criteria proposed by an international expert panel and was diagnosed in participants with hepatic steatosis by ultrasonography and one of the following criteria: overweight or obesity, type 2 diabetes mellitus, and metabolic dysregulation13. Participants with liver steatosis and regular alcohol consumption of > 30 g/day were diagnosed with ALD according to the reference data for males13.The diagnosis of ALD was based on the standard guidelines14.Alcohol consumption was assessed using a health questionnaire during the health checkups. The participants were asked the following question: “How many days per week do you consume alcohol on average?”  “How many cups of sake do you consume at one opportunity of drinking?” . A cup (180 mL) of sake corresponds to one-quarter bottle of wine . Daily alcohol consumption was calculated based on the frequency of alcohol consumption and amount of alcohol consumption during a single sitting, and participants with an alcohol intake of ≥ 20 g/day were assessed as having excessive alcohol intake based on the Japanese reference data13. The AUDIT consists of 10 questions on consumption (Q1–Q3), dependence symptoms (Q4–Q6), and alcohol-associated problems (Q7–Q10), and each question is scored from 0 to 4, as previously reported15. AUDIT-consumption (AUDIT-C) was assessed by the first three questions of AUDIT (Q1–Q3) as a shortened version13.The alcohol use disorders identification test (AUDIT) was administered to all participants as a self-reported questionnaire according to the guidelines of ALDU test. The Kruskal–Wallis test was used for multiple pairwise comparisons. Predictors of NAFLD, MAFLD, and excessive alcohol intake were assessed using logistic regression analysis and expressed as odds ratios (ORs) with 95% confidence intervals (CIs). Predictors with P values < 0.05 in the individual analysis were included in the multivariable analysis, considering the importance of variables and confounders. The discriminative ability of each variable was assessed using receiver operating characteristic (ROC) curve analysis and was presented as the area under the curve (AUC) with 95% CI. The optimal cutoff value was estimated using the Youden index value, and discriminative ability was shown by sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). The significance threshold was set at P < 0.05. Statistical analyses were performed using JMP version 16.2.0 software  and R version 4.1.2 software .Continuous variables are presented as mean and standard deviation. Categorical variables are presented as numbers and percentages (%). The two groups were compared using the chi-square test or Mann–Whitney All study procedures complied with the ethical requirements of the national and institutional committees that oversee human studies, and the 1964 Declaration of Helsinki and its later revisions. The study design was reviewed and approved by the Ethics Review Committee of the Graduate School of Medicine, Gifu University, Japan . The study design and objective were fully explained, and data were collected after obtaining written informed consent from all participants.2, respectively. Of the participants, 56 (18%) had hepatic steatosis and NAFLD, MAFLD, and ALD were diagnosed in 53 (17%), 34 (11%), and 3 (1%) of the participants, respectively  and AUDIT-C  scores than those without ALD. However, no significant difference was found in clinical variables, including biochemical analysis data, between the two groups (Table ALD was observed in three participants (1%), who had significantly higher AUDIT  and BMI  were independently associated with MAFLD. The ROC analysis showed that serum ALT levels and BMI had the larger AUC than other biochemical parameters for identifying MAFLD , BMI , and NAFLD  and AUDIT-C  scores had good discriminative ability to identify ALD with AUCs of 0.98 (95% CI 0.96–1.00) and 0.92 (95% CI 0.80–2.86), respectively , MAFLD (11%), ALD (1%), and excessive alcohol intake (4%) among the male graduate students. Japanese male young adults with NAFLD or MAFLD were mainly characterized by overweight/obesity and metabolic risk abnormalities, whereas none of them had type 2 diabetes. Furthermore, we demonstrated that serum ALT levels, BMI, and AUDIT are powerful screening parameters to identify high-risk male graduate students. This study, therefore, expanded our knowledge on the risks of CLD in Japanese male young adults and provided simple screening methods to identify students at risk for advanced CLD and severe outcomes.17. Furthermore, the prevalence of NAFLD in Japan is predicted to increase17. Despite the accumulated evidence of NAFLD prevalence, very few attempts have been made to determine at-risk individuals among young adults. The prevalence of NAFLD in our study (17%) seems reasonable, given that the Global Burden of Disease data have shown that the prevalence of NAFLD is approximately 9%, 16%, and 21% among ages 15–19, 20–24, and 25–29 years, respectively4. When comparing NAFLD with MAFLD in our study, the prevalence of MAFLD was lower than that of NAFLD among relatively young particpants18. However, MAFLD prevalence was higher than that of NAFLD in other studies with older participants19. Reviewing the results of our study with young participants and other studies with older participants, we assumed that metabolic changes due to aging are a strong driver of MAFLD and that the prevalence of MAFLD exceeds that of NAFLD around middle age. A large cohort study revealed that MAFLD, but not NAFLD, was associated with an increased risk of all-cause mortality8. Given the impact of MAFLD on outcomes, early detection and intervention of MAFLD, especially in young population, may play a critical role in preventing the future burden of CLD.As for the prevalence of NAFLD, a recent Japanese meta-analysis showed that the overall prevalence of NAFLD in Japan is 26% and males have a higher prevalence than females (34% vs. 16%)8, effective and convenient markers for MAFLD are urgently required. For large-scale screening of MAFLD, serum biomarkers are reasonable and preferred because imaging techniques are impractical in terms of cost effectiveness and availability11. The guidelines of NAFLD recommend the FIB-4 index or NAFLD fibrosis score, both of which comprise serum ALT levels, to stratify the risk of advanced fibrosis and avoid unnecessary liver biopsy11. Although these scoring methods are useful and well-validated for screening patients with advanced fibrosis, their use in health checkup setting is not yet common because platelet count and serum albumin levels are not mandatory for school health checkup including university. Therefore, there is a need for a simple screening method to detect NAFLD and MAFLD in early stage to offer an opportunity to prevent disease progression.As MAFLD has a stronger impact on mortality than NAFLD20. However, our study provides meaningful evidence that measurement of serum ALT levels is an easy and practical approach for screening MAFLD among male young adults. The results of our study also strengthen the importance of biochemical analysis to stratify the risk of CLD during health checkups, even among young adults.Our study revealed that a simple measurement of serum ALT levels may be sufficient to detect high-risk populations and is an ideal biomarker to identify NAFLD and MAFLD in terms of AUCs. We also found that the optimal cutoff value of ALT showed relatively high sensitivity and specificity for identifying MAFLD in this study. There are only a few reports on noninvasive biomarkers of MAFLD, which are poorly investigated compared to that of NAFLD. Although liver function tests have been the most traditional approach to investigate CLD, recent guidelines have concluded that this strategy lacks evidence for both screening and diagnostic methods and requires further investigation21. Since obesity plays an important role in the development of nonalcoholic steatohepatitis22, our results may reflect that the pathogenesis of MAFLD has a stronger impact on hepatocellular damage than that of NALFD. This can be a reasonable explanation as previous studies have shown that MAFLD has a worse survival rate than NAFLD8. Therefore, it is reasonable to focus on MAFLD as a target for screening and intervention in the health checkup setting.Another interesting finding in our study was that serum ALT levels were significantly higher in participants with overlapping conditions than in those with NAFLD only. Hepatocellular damage is the pathophysiological component of CLD and, therefore, serum ALT levels have a strong impact on liver-related and all-cause mortality2) and NAFLD (21.5 kg/m2) were lower than the cutoff value for obesity proposed by the Japan Society for the Study of Obesity (≥ 25 kg/m2)23. A recent study has suggested that even normal weight population can develop NAFLD because of fat accumulation and reduced muscle mass24. Since BMI strongly correlates with visceral adipose tissue23, our study suggests even nonobese male young adults can develop MAFLD and NAFLD, and BMI could be a robust marker to stratify the risk of these CLDs.In addition to serum ALT levels, BMI was also a robust marker to identify MAFLD and NAFLD among Japanese male young adults in terms of AUCs. Interestingly, the cutoff values of BMI to identify MAFLD (22.9 kg/m26. In addition, the prevalence of ALD in the Japanese population is around 2% based on a recent review27. In our study, approximately 4% of students consumed ≥ 20 g/day of alcohol, and 1% had ALD. A previous study of alcohol use in Japanese college students showed that 13% of students had excessive alcohol intake28. This discrepancy can be explained by the difference in region, academic degree, and decreased social opportunity for drinking because of coronavirus disease 2019. For screening, the AUDIT and AUDIT-C were the only methods used to identify the risk factors for cirrhosis. Ideally, all participants should be assessed by AUDIT for ALD screening; however, our data show that the AUDIT-C, a shortened AUDIT, can be a reasonable alternative considering the limited time available to complete health checkups. Accumulated evidence has shown that a high proportion of patients with ALD do not exhibit any clinical or biochemical abnormalities13, and our study also confirmed that there is no difference in biochemical parameters between participants with and without ALD in Japanese male young adults. Therefore, identifying excessive alcohol intake is a fundamental step for screening ALD. A recent Japanese nationwide survey revealed that cases of liver cirrhosis associated with ALD is increasing and, therefore, it is essential to identify alcohol misuse and ALD among young adults to reduce the burden of CLD29. This report contains the first evidence of the usefulness of AUDIT and AUDIT-C for screening ALD among Japanese young adults.The prevalence of and screening methods for alcohol misuse and ALD among young male adults were investigated in our study. In general, the prevalence of excessive alcohol intake depends on the definition and cohort, and ranges from 10 to 40% in the Japanese population21. In addition, ultrasonography is costly and requires well-trained examiner. Given that very few attempts have been made on the screening of CLD in younger population, we believe that our findings have meaningful implication as using health checkups to assess and inform individuals about their risk for CLD may help them to make lifestyle modification that reduces the future burden of CLD.The clinical relevance of our findings should be explained. We found that health checkup is an important opportunity to identify, MAFLD, NAFLD, and ALD in Japanese male young adults. However, it is impractical to provide liver ultrasonography to all individuals based on our cutoff values because the values were relatively low considering the commonly used cutoff values30. Second, liver biopsy was not performed in this study and liver steatosis was diagnosed only by ultrasonography. The small number of ALD cases may have limited the statistical power of our study. Third, we lacked data on well-validated scores, such as FIB-4 index and NAFLD fibrosis score, and future studies should evaluate the clinical relevance of these scores in younger populations. Therefore, a multicenter study that includes both males and females is required to validate our results. Despite these limitations, this study may have important implications for screening and identifying the prevalence of MAFLD and excessive alcohol intake among male young adults in a health checkup setting.This study had several limitations. First, this was a single-center study that included only males and our results may not be applicable to other regions and females. Especially, females were initially excluded from the study considering the sex differences of obesity, type 2 diabetes, other metabolic abnormalities, and normative values of liver enzymesIn conclusion, our study provides the first evidence of the prevalence of MAFLD and ALD in young Japanese male adults. Furthermore, simple measurements of serum ALT levels, BMI, AUDIC, and AUDIT-C are useful for screening CLDs. Our study may guide the establishment of strategies for MAFLD and ALD identification in younger generations, and confirm the importance of health checkups for early detection and intervention in reducing the future burden of CLD.Supplementary Information.


Researches on the effect of hemodynamic stabilization on the implantation of conservative management for pediatric high-grade renal traumas are lacking. We aimed to assess the effect of maintaining the initial hemodynamic stability of pediatric patients with grades 3–5 renal trauma on the implementation of the conservative treatment and identify its defining factors.A prospective study was performed on pediatric patients with grade 3–5 renal traumas who presented to our hospital during July 2020–June 2022. Hemodynamically stable patients were compared with the unstable patients for clinical characteristics, hemodynamic stabilization, and rates of success of conservative treatment.Forty-three patients were studied, including 26 boys and 17 girls. Of them, 28 (65.1%) patients presented with hemodynamic stability and 15 (34.9%) patients were unstable. Overall, 32 (74.4%) patients achieved and/or maintained hemodynamic stability for conservative management. There was a significant difference in blood pressure level at presentation (p < 0.001). The improvement of the hemodynamic parameters was significant per group and, in comparison (p < 0.001). The size of hematoma was significantly smaller in patients with hemodynamic stability (p = 0.023). Despite the longer (p = 0.033) hospital stay with conservative management, the rates of blood transfusion (p = 0.597) and hospital stay (p = 0.785) were not significantly different between both groups. The rates of nephrectomy and mortality were 14% and 0%, respectively. Blood pressure was independently associated with the achievement of maintained hemodynamic stability for conservative management (p = 0.022).Hemodynamic stabilization seems to be effective and safe for implementing successful conservative management for pediatric patients with high-grade renal traumas. Blood pressure was the only independent factor of maintaining hemodynamic stability. The rates of surgical intervention and complications are associated with the high grades of renal trauma in pediatrics , 2. ReceThis study hypothesized that achieving and maintaining initial hemodynamic stability provides higher rates of implementation and success for conservative treatment. The aims were to assess the effect of maintaining hemodynamic stability during the first 24 h on the implementation of conservative management and its predictors in pediatric patients with grades 3–5 renal trauma. Hemodynamic stability was defined as the control, correction, or resolution of the clinical and laboratory manifestations of hemodynamic instability for 24 h. The primary outcomes were the rates of hemodynamic stability within the first 24 h and successful conservative management. However, the secondary outcomes were the rates of nephrectomy and mortality.A prospective study was conducted on pediatric patients with grade 3–5 renal traumas treated in our hospital during July 2020–June 2022. This study was approved by the ethical committee at our university. The inclusion criteria were patients aged < 18 years with renal trauma grades 3–5. Patients who had been explored by trauma surgeons without proper urological assessment, initially managed in another hospital before referral, or had trauma older than 24 h were excluded from the study.The clinical workups included stabilizing and monitoring the vital signs  and hematuria or perinephric hematoma with intravenous fluids  were administered before blood transfusion (packed red blood cells in a dose of 10 ml/kg up to 4 times till resuscitation was achieved). The initial laboratory workups included the hematocrit, hemoglobin levels, and serum creatinine. Hemodynamically stable patients, either from the start or after initial hemodynamic resuscitations, were managed by the conservative approach, while patients with persistent hemodynamic instability were managed by immediate surgical exploration.In the conservative approach, patients were managed by observation and monitoring of their vital signs, hemodynamic status, and laboratory values. Further laboratory tests included urine analysis, random blood sugar, and blood gases when indicated. Medications and serial examination, testing, and reimaging were performed while the surgical team and the patient were ready for urgent potential surgical interventions. In the interventional approach, patients were managed by minimally invasive maneuvers or open surgery. These were the components of management in our hospital . Our proAccording to their hemodynamic status at presentation, patients were classified into two groups. The first group included patients who were hemodynamically stable (Stable group), and the second group included patients who were hemodynamically unstable from the start (Unstable group). Respective to these groups, patients who were stable from the start (from the Stable group) and those who were stabilized and maintained (from the Unstable group) on hemodynamic stability for the first 24 h after trauma were allocated to receive conservative management. However, immediate surgical exploration with renorrhaphy, partial, or total nephrectomy was implemented to the unstable patients in both groups: First, patients from the Stable group who became unstable . Second, patients from the Unstable group who failed to be stabilized or failed to maintain robust stabilization after resuscitation (persistent instability).Regardless of the trauma grade, conservative treatment included complete bed rest, broad-spectrum antibiotic, hydration, analgesic, serial monitoring of the hemodynamics, vital signs, urine color, serial hemoglobin and hematocrit values, and reimaging in the form of serial abdominal ultrasonography follow-up for the size of hematoma in all cases. In addition, CECT was repeated at 3-month duration from the date of trauma in patients with hematoma seen by abdominal ultrasonography at 1-month follow-up. Patients stayed in the hospital for strict monitoring until there were stable vital signs, clear urine, regression or absence of perinephric hematoma. After discharge, each patient had a follow-up for three months. At each visit, a history of recurring hematuria, loin pain, or fever is taken with follow-up ultrasonography for tracing of the residual hematoma.Postoperative follow-up was performed with strict observation of the vital signs. Further evaluations were carried out by abdominal ultrasonography and serial measurement of hemoglobin and hematocrit values up to discharge from the hospital.The statistical package for social sciences, version 20.0  was used to analyze the data. In descriptive analyses, continuous variables were presented as mean ± standard deviation or median and range. However, categorical variables were presented as the number and percentage of each category. The continuous variables were compared with Student t-test or Mann Whitney U test, according to data distribution. However, the categorical variables were compared with the chi-squared or Fisher’s exact test accordingly. A multivariate logistic regression was performed to assess the factors contributing to the maintenance of hemodynamic stability. Two-tailed p < 0.05 was considered as statistically significant.During the duration of this study, 48 children were managed in the Trauma Unit, Assiut University Hospital, for grade 3–5 renal traumas. Of them, 5 patients did not fulfill the inclusion criteria and were excluded from the study; two patients were explored in another center before presentation to our hospital, and three patients were explored immediately with an inaccurate grading of renal trauma due to hemodynamic instability caused by multiple organ traumas Fig. .2. They included 26 boys (60.5%) and 17 girls (39.5%).Forty-three eligible patients were included in the current study. Their mean age was 9.5 ± 4.6 years  and mean body mass index was 24.4 ± 5.3 kg/mThe first group included 28 65.1%) patients with hemodynamic stability, and the second group included 15 (34.9%) patients with hemodynamic instability. The demographic and clinical characteristics of both groups at presentation to the emergency unit are presented in Table % patientDuring the first 24 h and through the whole first week after trauma, the changes in blood pressure were demonstrated Table .Of the 43 patients, 32 (74.4%) patients achieved and maintained hemodynamic stability in the first 24 h and they successfully completed conservative management. However, 11 25.5%) patients were hemodynamically unstable or failed to maintain robust stability and they were managed by surgical interventions. In the unstable group, nine patients received successful conservative treatment. Among them, grades of trauma were 3, 4, and 5 in two, three, and four patients, respectively . However, the length of hospital stay was significantly longer with the conservative management (p = 0.033) than with the interventional management Table .In a multivariate analysis, blood pressure was the only independent factor of maintaining hemodynamic stability for implementing conservative management (p = 0.022) Table .A large body of research has been conducted to verify the efficacy and safety of management approaches of high-grade pediatric renal traumas so far , 5, 9. DFurthermore, conservative management may be followed by a potential relative decline in renal function . This efTo make a decision about conservation in children with major renal trauma, there should be contingent safety and feasibility criteria available to guarantee timely intervention. In addition, this warrants the availability of a full range of flexibility in equipment and manpower for urgent exploration of the patient, whenever the indication for surgical intervention supervenes. The most critical period is the time from the presentation of the patient to the time of decision making, when the challenges of stabilizing a patient with a major hemorrhage are at their maximum , 4, 10. The mechanism of high-grade renal trauma is usually blunt injury, and grades 3 and 4 represent the highest proportion in those patients . In addiThe recent studies showed high overall success rates for the conservative approach, reporting rates up to 92.5% [3.7]. The current results showed a slightly lower rate, but they are still relatively high in these grades of trauma in the context of the literature [Extended renal investigations may not be required if the child’s hemodynamic instability persists or if there is no response to blood transfusions that are up to 3 units because immediate exploration is absolutely indicated. Expanding or pulsatile perirenal hematomas represent another indication for surgical therapy. In addition, the other common indications include significant urine extravasation, extensive (> 20%) nonviable tissue, arterial damage, and insufficient staging . The surPartial nephrectomy is a higher step in the surgical repair of high-grade renal traumas with devitalized tissues. It is indicated in cases of a completely shattered pole of the kidney that is ischemic and its arterial supply is beyond repair. Early surgical debridement is the best treatment for devitalized renal parenchyma. Intraoperative signs of a devascularized pole or segment of the kidney include complete separation or bluish discoloration of the suspected tissues .There are many indications for total or simple nephrectomy of the injured kidney. They include grade 5 injuries that are deemed irreparable, such as major vascular injuries, a shattered kidney, multiple concurrent injuries, and uncontrolled hemorrhage , 13. NepThe rate of nephrectomy is one of the main outcomes of the management of high-grade renal traumas. Compared to adult trauma, juvenile trauma has a lower nephrectomy rate . It may The minimally invasive interventions in pediatric patients include angioembolization techniques, ureteral stents placement or percutaneous drainage of the obstructed kidneys , 19, 20.As a prospective study, our study may contribute to filling the gap in research on the hemodynamic effect on management by providing information about the decision-making in the management of pediatric high-grade renal traumas. In addition, it allowed patient selection criteria that helped recruit the patients and manage them without harmful effects from the application of the conservative approach.Limitations of his study included the non-randomized allocation of patients to the approach of management. The low incidence of high-grade renal trauma in pediatrics was a cause of the difficulty in recruiting a relatively larger sample size. In addition, this small sample size hindered studying the effect of hemodynamic stability on management in each grade of trauma. Furthermore, the short-term follow-up and evaluation of the kidneys did not allow us to know the extent of effect of trauma on the functions of the preserved kidneys after these high-grade traumas.In high percentages of patients presented with grades 3–5 renal trauma, achievement and maintenance of robust hemodynamic stability during the first 24 h were feasible, even in the unstable patients. It enhanced the implementation of conservative management and provided a high success rate and relatively low rates of nephrectomy and mortality. In addition, the blood pressure level at presentation was an independent factor in maintaining hemodynamic stability sufficient for successful conservative management in these patients.


Conversely, oil and protein content were inversely correlated in most of yielded mutant lines. Together, those findings introduce novel soybean lines with favorable agronomic traits for the market. In addition, our research sheds light on the value of using gamma rays treatment in enhancing genetic variability in soybean and improving oil, protein contents and seed yield.Mutation breeding is one of the effective techniques used for improving desired traits such as yield quality and quantity in economic crops. The present study aims to develop oil and protein contents in addition to high yield attributes in soybean using gamma rays as a mutagen. Seeds of the soybean genotypes Giza 21, Giza 22, Giza 82, Giza 83 and 117 were treated with gamma rays doses 50, 100, 200 and 300 Gy. Plants were then scored based on morphological parameters correlated with yield quantity including plant height, seed weight and valuable protein and oil contents. Mutant lines exhibiting the highest yield attributes were selected and used as parents for M2 generation. The M2 progeny was further assessed based on their ability to maintain their yield attributes. Twenty mutant lines were selected and used as M3 lines. The yield parameters inferred a positive effect of gamma irradiation on the collected M3 mutant lines compared to their parental genotypes. 100 Gy of gamma rays gave the highest effect on the number of pods, branches and seeds per plant in addition to protein content, while 200 Gy was more effective in increasing plant height, number of pods per plant, and oil content. Six mutant lines scored the highest yield parameters. Further assessment inferred an inverse relationship between oil and protein content in most of the tested cultivars with high agronomic features. However, four mutant lines recorded high content of oil and protein besides their high seed yield as well, which elect them as potential candidates for large-scale evaluation. The correlation among examined parameters was further confirmed  Glycine max L. 2n = 2x = 40), a versatile and nutritious legume, holds immense value as a staple in the realms of human food, livestock feed, and oil production  alongside promising advanced line (Line 117) were used in this study. The utilized genotypes were obtained from the Legume Crops Research Department, Institute of Field Crops, ARC, Giza, Egypt .60 source . These doses were selected based on the germination test conducted prior to the current study in which we considered the 50 Gy and the 300 Gy as the minimum and the maximum doses with least inhibition/most germination. The dose was applied at the rate of 5.6 Gy/minute.The seeds of the soybean cultivars were treated with the following doses of gamma rays 50, 100, 200 and 300 Gy with the radioisotope Co2O5 ha−1 as superphosphate (15.5% P2O5) and 100 kg K2O ha−1 as potassium sulfate (48% K2O). Nitrogen fertilizer was applied in the form of ammonium sulfate (20.5 N%) at a rate of 70 kg N ha−1 in two equal doses before the first and second irrigations. The seeds were inoculated with the microbial symbiont Rhizobium japonicum immediately before sowing. The field trials were irrigated using surface irrigation with 10 days intervals.The irradiated seeds of soybean cultivars (200 seeds for each cultivar) were grown independently in rows at the end of May of each growing season. Non-treated seeds were also grown at individual lines to serve as a control treatment. The assessed genotypes were sown in three replicates using Randomized Complete Block Design (RCBD). Each genotype was sown in 6-m long row with 0.65-m wide and 0.2-m between hills. Each hill was sown with four seeds and thinned to two seedlings after full emergence. Phosphorus and potassium fertilizer were added during soil preparation at a rate of 100 kg PThe M2 plants were individually screened for morphological changes for further selection. The selected mutants were sorted out based on the following parameters: dwarf (D), semi-dwarf (SD), high number of pods (HNP), high number of seeds (HNS), heavy seeds (HS), height of first pod (HFP), long stem (LS), small seeds (SS), medium seeds (MS) and seed shape (SP).The selected M2 mutant lines were sown to obtain the M3 generation. The M3 seeds were grown in three replications with 20 seeds per replicate in a randomized complete block design. The growing distance was 65 cm between rows and 20 cm between seeds. Data were collected at the seed maturity stage. The collected data included the previously described morphological characters besides the oil content and the total protein content in each replicate.To determine the % soybean oil, the soybean seeds were cleaned and ground by Moulinex blender Type 716 (France) to pass through a 1 mm sieve. The powder was defatted by hexane in a Soxhlet extractor . The totpost hoc test was further used to define significance among treatments at the probability level 5% (P < 0.05). The statistical analyses were performed using R statistical software version 4.1.1.The collected data were statistically analyzed using the analysis of variance (ANOVA) method, and Tukey’s HSD The mutagenicity of gamma radiation was tested on plants from M2 populations. Different promising derivative mutants were identified in M2 populations of the mutated five soybean genotypes. These mutants involved dwarf, semi-dwarf, a high number of pods, a high number of seeds, heavy seeds, the height of the first pod, long stem, small seeds, medium seeds and seed shape . Screenip < 0.001) among the derived mutants for all evaluated traits at levels (p < 0.001), while the differences among the three replicates were nonsignificant (p > 0.05) for all traits  was conducted to explain the relationship among different traits of the soybean M3 mutants and their corresponding parents. The PC1 and PC2 explored the most variance presenting 80.99% , and were used to construct the PC-biplot . The PC1These findings are valuable for plant breeders to build selection criteria on strong positively associated characters. Likewise, the heatmap and hierarchical clustering based on the yield traits, oil content and protein content divided the evaluated genotypes into distinct clusters . The genIn soybean breeding, development of new cultivars with improvement characters such as yield, oil and protein contents is considered the main objective. The influences of gamma radiation on certain plants and seed properties have been investigated . In thisThe exposure to gamma radiation can be used to induce mutations and thereby generate genetic variation from which desired mutants may be selected . MutatioAccording to Protein content trait increased in the H117-HNP-1 mutant using a 200 Gy gamma irradiation dose. The highest reduction in the protein content (−0.07) was achieved in the G82-D-1 mutant by the use of 100 Gy with the Giza 82 genotype. Previous studies that used gamma irradiation with several plant species reported an increase in oil content which support our results . ConversClustering analysis based on yield, oil content, and protein content traits grouped the 25 soybean genotypes  into four, five and five different clusters, respectively. This indicates that the 25 soybean genotypes displayed a wide range of diversity in terms of yield, oil content, and protein content traits. This clustering will facilitate the selection of parents for a specific trait in hybridization programs. Several studies reported the use of clustering analysis to group the soybean genotypes into different clusters . The denThis investigation will support conventional breeding experiments  to improThe current investigation reported high levels of phenotypic variations for the investigated traits among the twenty soybean mutants and their corresponding parents. These mutant lines could be candidates for further genetic improvement of soybean genotypes in breeding programs. Among the studied traits, a positive correlation was observed between protein content and plant height, and between the 100 seeds’ weight and oil content. Therefore, effective phenotypic selection could be applied to the genetic improvement of soybean by considering these favorable attributes together. The majority of the studied traits revealed positive associations with each other, which will allow the combined improvement of these traits by selecting only easily measurable and highly heritable characters. Cluster analysis using investigated traits grouped the 20 soybean mutants and five parent genotypes into four, five and five different clusters based on yield and contributed traits, oil content and protein content, respectively.10.7717/peerj.16395/supp-1Supplemental Information 1Mean performance data for studied traitsClick here for additional data file.