Thecomposition of dynamic covalent imine libraries (DCL) adaptsto the presence of the hexameric resorcinarene capsule. In the presenceof the self-assembled capsule, a kinetic and thermodynamic modulationof the imine constituents of the DCLs was observed, which was inducedby an unusual predatory action of the capsule on specific imine constituents.More complex 2 × 2 DCLs also adapt to the presence of the hexamericcapsule, showing a thermodynamic and kinetic modulation of the constituentsinduced by the predatory action of the capsule. By cross-referencingexperimental data, a good selectivity (up to 66%) for one constituentcan be induced in a 2 × 2 DCL. Onthis basis, one of the aims of enzyme mimicry3 is the synthesis of artificial systems able to work selectivelyin the presence of a complex mixture of reagents.Nature is a continualsource of inspiration for those scientistsinterested in mimicking the strict level of selectivity and efficiencythat are the basis of living systems.6 has arouseda particular interest. In DCC, simple building blocksare held together by reversible covalent bonds to form a library ofproducts that, under thermodynamic equilibrium conditions, are continuouslyinterconverting.6 Under these conditions,the library is usually able to respond to an external stimulus stimulus 1 by chan11 Formationof an imine bond is a dynamic process where a carbonyl compound reactsin a reversible manner with an amino group with the loss of water the internalπ···electron-rich cavity of 1375 Å3 is able to recognize neutral and cationic species and to stabilizetransition states, due to secondary interactions; (b) the capsule CR6 behaves as a mild Brønstedacid with a pKa value of about 5.5–6.0;18a (c) four bridging water molecules show a H-bond-donatingfree valence, which is catalytically relevant.19 In addition, previously reported data20 show that the CR6 capsule isable to exert a substrate selectivity, whereas stereo- and regioselectivitytoward the products are also generally observed.Recently, in biomimicry, much attentionhas been devoted to catalyticprocesses in a nanoconfined space using self-assembled capsules.sule CR6 2 has becsule CR6 2 in the CR6,21 exploiting its mildacidity and its ability to stabilize cationic intermediates and transitionstates. Successively, our group reported experimental and computationalevidence of the formation of an iminium specie inside CR6.22Tiefenbacher and co-workers reported the first example of catalysisof formation of an iminium group inside CR6. As stated by Lehn,7a “changesin expression of the different constituents as a factor of externalparameters represent an adaptation of the system to environmentalconditions”. On this basis, we wonder if the composition ofdynamic imine libraries adapts to the presence of CR6, which, in addition to catalytic abilities,usually also shows a substrate and product selectivity.Theseconsiderations prompted us to investigate the behavior ofdynamic imine libraries in the presence of A2a and A2b in single experiments (CR6.We start thisstudy by investigating the formation of imines eriments 1 in the 2a and p-chloroaniline A were mixed in an equimolarratio (42.3 mM) in water-saturatedCDCl3 at 30 °C in the presence of capsule CR6 (1 equiv), the formation of imine A2a was detected in the reaction mixture after 30 min  spectroscopyusing 1,1,2,2-tetracloroethane (TCE) as an internal standard. Aliquotsof the reaction mixtures were added to DMSO (Supporting Information) in order to disaggregate CR6, and the imine signals were integrated with respectto the signal of TCE. In the absence of the CR6 capsule, two imine constituents, A2a and A2b, were formed in equal quantities up to 48 h . The results in A2a was kinetically favored, whereas A2b wasthe thermodynamic product under these conditions.Imine 2a and p-chloroaniline A were reacted in the presenceof CR6 in order to form onlyimine A2a. After 24 h, A2a was formed in34% yield CR6 acts as a catalyst by acceleratingthe formation of imineconstituents A2a and A2b, due to its mildacidity and capability to stabilize cationic intermediates.(b)CR6 acts as an external stimulus because the DCL compositionof A2a and A2b adapts to its presence. Theformationof imine A2a is initially favored, whereas A2b prevails at longer time. Thus, under these conditions, A2a and A2b represent the kinetic and the thermodynamicadducts, respectively.The evolution of the imine composition in 23 In detail, a competition experiment was carriedout in which benzaldehyde 2a and p-trifluoromethylbenzaldehyde 2b were in competition to occupy the inner cavity of CR6. The uptake of 2a/2b inside CR6 wasmeasured by quantitative 1H NMR experiments, in which thealdehydes 2a and 2b (42.3 mM each one) weremixed in the presence of 1 equiv of CR6 in water-saturated CDCl3. The quantity of encapsulatedaldehyde was obtained by determining the difference between its initialconcentration and the concentration of the free aldehyde in solution.The 1H NMR signal of the free aldehyde was integrated withrespect to the signal of the internal standard (TCE). After equilibration,a 52% uptake of benzaldehyde 2a inside CR6 was measured, a value significantly higherthan that obtained for the aldehyde 2b (5%). Thus thehexameric capsule CR6 shows ahigher affinity for benzaldehyde 2a with respect to p-CF3-benzaldehyde 2b. Clearly,this result is in accord with the finding that A2a ispreferentially formed in the early stage of the reaction, where thecapsule is filled to a greater extent with benzaldehyde 2a.In order to getmore insights on the mechanism of this kineticand thermodynamic modulation of the DCL in 2a inside CR6 was obtained by1D and 2D NMRstudies and, in particular, by HSQC experiments . From thesestudies, it emerges that the benzaldehyde is encapsulated inside CR6 with slow kinetics with respectto the NMR time scale (600 MHz). Analogously, the encapsulation ofthe aldehyde 2b inside CR6 was studied by 1D and 2D NMR experiments . Analogousstudies were reported in the Supporting Information in order to show the encapsulation of p-chloroaniline A inside CR6 .Proof of the encapsulation of benzaldehyde A2a and A2b remains to be understood. In detail, we wonder why A2b prevails for a long time whereas A2a decreases withrespect to its initial percentage.The fate of the two imines A2a wasdissolved in water-saturated CDCl3 solution in the presenceof CR6 (1 equiv), after 30 min,62% of A2a was hydrolyzedto 2a and A . These data stronglyindicate that the hydrolysis of A2a occurs inside CR6 due to its catalytic activity.This was confirmed by the finding that, in the presence of DMSO, asolvent able to break down the capsule,15 no conversion of A2a into 2a and A was observed. In contrast, the hydrolysis of A2b to 2b and A in the presence of CR6 was slower . In silico calculations were in accord with these results(Supporting Information). Quantum-mechanical calculations(Supporting Information) indicate an enthalpicstabilization of −22.14 kcal/mol and a Gibbs free energy stabilizationof −8.08 kcal/mol for the formation of the A2a⊂CR625 complex. However, the formation of the complex A2b⊂CR6 is unfavored inenthaplic as well as Gibbs free energy terms.25On this basis, we can explain the origin of thekinetic and thermodynamicmodulation of the DCL in y of CR6 5–8, whic capsule 8, cataly26 and noncovalentinteraction (NCI)27 (see Supporting Information) analyses were performed on complexes A2a⊂CR6 and A2b⊂CR6 to identifythe second-order interactions between the capsule and the imine. Second-orderperturbation theory (SOPT) analysis of the FOCK matrix in NBO basisclarified that the better binding affinity of A2a isprincipally due to a strong hydrogen bonding interaction . Regardingthe A2b⊂CR6 complex, because of the steric demand imposed by the trifluoromethylgroup, A2b is forced to stay on the axis that joins twovertexes of the capsule eraction 7e betweef 2.81 Å 7e and a  capsule 7c,d, witcrocycle 7c,d. In 29 of thecatalyst (CR6) on one of theconstituents (A2a) plays a crucial role.In summary, these results show thatthis is a rare example of kineticand thermodynamic adaptation of a DCL, in which the intraspecific“predatory” effectA2a and A2b in the presenceof lower quantities of “predator” CR6 (CR6 (0.5 equiv), the kineticallyfavored imine A2a was prevalent up to 2 h (CR6 (0.5 h). Under these conditions, the thermodynamicimine A2b began to prevail at 4 h, and finally, the quantityof A2a after 48 h was slightly higher than that obtainedin the presence of 1 equiv of the capsule  and 1.0equiv (15%) of 6CR. This result clearly indicatesthat the stability of the imine A2a in the DCL increasesby decreasing the quantity of capsule CR6, showing in this way its predatory effect on A2a.Now, inorder to corroborate this assumption, we studied the distributionof the constituents tor” CR6 9. When tp to 2 h 9, a timesule see 9. Decreap-nitrobenzaldehyde 2c was used instead of 2b, the DCL of the components A/2a/2c showed an analogous behavior(see Supporting Information). In detail,in the presence of CR6, an adaptationof constituents was thermodynamically driven by the hexameric capsuletoward the imine A2c derived by aldehyde bearing an electron-withdrawinggroup on the phenyl ring, whereas the constituent A2a remained the kinetically favored one .Interestingly, when the p-OMe-benzaldehyde 2d was used together with 2a and A as a component of the DCL, the formation of imines A2a and A2d was observed in very low yields inthe presenceof CR6.In contrast, when the A2a was favoredover time .Interestingly,in this case, imine B and aldehydes 2a and 2b (R = CF3)  4 as compr mixing 10b, wherr mixing 10a. Wither 0.5 h 10b. AfteB2a in the experimentin CR6 on the imine B2a. Encouraged by thishypothesis, we evaluated the stability of B2a in water-saturatedCDCl3 in the presence or in the absence of capsule CR6  higher than thatof A and B, and aldehydes 2a/2b in CR6 , is not protonated by CR6 in wet CDCl3 in the presence or in theabsence of CR6. As reported in Supporting Information  and CR6, imines E2a and A2a were formedin 50 and 25% yield, respectively  and B (p-H)  7, and weA2a and B2b were the main components, followed by B2a and A2b in a distribution of 25, 20, 17, and 11%, respectively, the kinetically favored imine A2a survived longer, thus also in this case, the predatoryeffect of the capsule toward A2a was suppressed .When the reaction was performed without the capsule,the compositionof the library showed no substantial preference for the distributionof the components 15a. When3 to work selectivelyin the presence of a complexmixture of reagents, the results reported in CR6 is able to work selectively inthe presence of complex mixtures of substrates, due to a fine controlof the encapsulated species, leading to the selective formation ofspecific imines. In order to further corroborate this result, we performeda new 2 × 2 experiment, changing the amine and aldehyde components.In accordance with one ofthe aims of enzyme mimicry,2d/C) and an electron-withdrawing trifluoromethylgroup (2b/D)  8. As in  capsule 16.C2b and C2d from p-methoxyaniline C were detectedas the main components of the mixture in a C2b/C2d ratio of 47/19, whereas imines D2b and D2d, obtained from the less reactive p-trifluoromethylaniline D, were present in almost negligible quantities. Over time,an increase in the quantity of C2b was observed, whichafter 24 h was the most abundant constituent of the mixture, witha composition of C2b, D2b, C2d, and D2d of 66, 12, 5, and 5%, respectively. In summary,by cross-referencing the data in p-OMe-benzaldehyde 2d or p-OMe-aniline C are present as components.After 0.5 h, imine constituents CR6. The DCL of imines A2a and A2b formed by benzaldehyde 2a, p-CF3-benzaldehyde 2b, and p-chloroaniline A adapts its composition in the presenceof CR6, showing a kinetic andthermodynamic preference of the constituents. In particular, the kineticallyfavored constituent A2a, obtained from benzaldehyde 2a, is preferentially formed immediately after mixing, dueto the preferred inclusion of 2a inside CR6. Surprisingly, the capsule shows a predatorybehavior toward imine A2a, which is quickly hydrolyzedto components A and 2a inside the capsule.On the other hand, imine constituent A2b, obtained from p-CF3-benzaldehyde 2b, is hydrolyzedslower than A2a. Uptake studies show that, after thehydrolysis of imine A2a, the benzaldehyde component 2a remains included in the capsule CR6. Interestingly, the hexameric capsule CR6 shows an analogous predatory action onother benzaldehyde-based imines such as B2a and C2a derived from aniline B and p-OMe-aniline C, respectively. Finally, more complexesof 2 × 2 DCL systems adapt to the presence of the hexameric capsule,showing a thermodynamic and kinetic modulation of the constituentsand leading to a good selectivity (up to 66%) for one of them.In conclusion, we have demonstrated that dynamiccovalent librariesof imine constituents are able to adapt their composition in responseto the presence of the hexameric resorcinarene capsule


The glymphatic system plays an important role in clearing the amyloid-β (Aβ) and tau proteins that are closely linked to Alzheimer disease (AD) pathology. Glymphatic clearance, as well as Aβ accumulation, is highly dependent on sleep, but the sleep-dependent driving forces behind cerebrospinal fluid (CSF) movements essential to the glymphatic flux remain largely unclear. Recent studies have reported that widespread, high-amplitude spontaneous brain activations in the drowsy state and during sleep, which are shown as large global signal peaks in resting-state functional magnetic resonance imaging (rsfMRI), are coupled with CSF movements, suggesting their potential link to glymphatic flux and metabolite clearance. By analyzing multimodal data from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) project, here we showed that the coupling between the global fMRI signal and CSF influx is correlated with AD-related pathology, including various risk factors for AD, the severity of AD-related diseases, the cortical Aβ level, and cognitive decline over a 2-year follow-up. These results provide critical initial evidence for involvement of sleep-dependent global brain activity, as well as the associated physiological modulations, in the clearance of AD-related brain waste. This study reveals strong coupling between the global fMRI signal and cerebrospinal fluid influx, finding that this is correlated with Alzheimer’s disease-related pathology, disease severity, and cognitive decline. This supports a link between spontaneous low-frequency brain dynamics and Alzheimer’s disease pathology, presumably due to their role in glymphatic clearance. The pathogenesis of Alzheimer disease (AD) is widely believed to be driven by the aggregation of toxic proteins, e.g., the amyloid-β (Aβ) and tau, which cannot be adequately cleared from the brain –3. The “The glymphatic process may be related to sleep-dependent global brain activity recently observed with resting-state functional magnetic resonance imaging (rsfMRI) –26. A piTo test this hypothesis, we examine multimodal data from the Alzheimer’s Disease Neuroimaging Initiative (ADNI)  with a fWe used imaging and behavioral data from 118 participants in the ADNI project. The sample included 7 AD patients, 62 mild cognitive impairment (MCI) patients, 18 significant memory concern (SMC) patients, and 31 healthy controls (HCs)  at the lag of +3 seconds , also consistent with the previous finding  (version: 07_28_19)”  (version: 07_28_19)”  . We transformed the gray matter mask from the MNI-152 space back to the original space of each participant/session to avoid spatial blurring from the registration process with the same rationale to a previous study [We extracted the mean BOLD signal from the gray matter regions of the cortex and regarded it as the global BOLD signal following the practice of previous studies by others ,77. A maus study . For eacThe CSF signals were extracted from the bottom slices of fMRI acquisition, which are near the bottom of the cerebellum for all the participants, to obtain high sensitivity to the CSF inflow effect as described previously . The fMRp-value for the BOLD–CSF correlations at different lags was obtained by comparing them against the null distribution.We calculated the cross-correlation functions between the global BOLD signal and the CSF signal obtained through the above procedures to quantify their coupling, similarly to the previous study . The croN = 158), and it was also compared across different subgroups, e.g., males versus females, using a linear mixed effect model. For the disease condition, we also tested whether the BOLD–CSF coupling strength (age and gender adjusted) changed with the severity of the AD-related symptoms (see details about the severity and staging of 4 conditions at http://adni.loni.usc.edu/study-design/). For APOE gene, we tested the relationship between the BOLD–CSF coupling strength (age and gender adjusted) and the number of APOE ε4 alleles. We repeated the same analysis for the augmented dataset with more AD patients and APOE ε4 alleles carriers.We quantified the BOLD–CSF coupling strength using their cross-correlation at the lag of +3 seconds (the negative peak of the cross-correlation function). We then related the BOLD–CSF coupling to age, gender, disease condition, and APOE gene. The association between the BOLD–CSF coupling and age was investigated across sessions  of fMRI image sessions we used were collected by ADNI 2, there are data from ADNI 3 and ADNI GO. These different studies may use slightly different image parameters and/or experimental setups. To make sure that our major findings were not caused by any potential differences, we examined the associations between the BOLD–CSF coupling and AD pathologies using only the data from ADNI 2. We further included the study cohort  as a covariate and retested these associations.Since the arousal fluctuation is a major confounding factor in rsfMRI ,88,89 anWe also repeated the above analysis in t test for continuous measures  and Fisher exact test for categorical measures  across groups . Significant differences (p < 0.05) for each comparison were marked with a bold format. The significance of the BOLD–CSF correlations was tested with a permutation method that shuffled the correspondence between the global BOLD signal and the CSF signal to build the null distributions for the BOLD–CSF correlation at different time lags, similar to the previous study [p-values were computed using the R package “lmerTest” based on the Satterthwaite degrees of freedom method [p-value less than 0.05 was regarded as statistical significance.Group comparisons of our sample characteristics  were perus study . Spearmaus study . Becauseus study . The corm method . We alsoS1 FigA) When we increased the sample size of AD and HC session (N = 199), the BOLD–CSF coupling shows a significant correlation with age across sessions . AD, MCI, SMC, and HC sessions are colored with blue, light gray, dark gray, and orange, respectively. Each dot represents a session. (B) Male participants have larger amplitudes of this BOLD–CSF coupling compared with female ones (p = 0.038) in this augmented dataset. (C) The strength of the BOLD–CSF coupling, after adjusting the age and gender effects, also decreases gradually (p = 0.005) along with the increased severity of disease condition, i.e., the axis of HC–SMC–MCI–AD. Importantly, significant differences can be found not only between the HC and MCI groups (p = 0.045) but also between the HC and AD sessions (p = 0.0078). (D) The age- and gender-adjusted BOLD–CSF coupling is also significantly correlated with the APOE ε4 allele number  across augmented sessions. Error bar in this figure represents the SEM. The underlying data can be found in ((TIF)Click here for additional data file.S2 FigA) The strength of the BOLD–CSF coupling adjusted for the disease condition  shows a significant correlation  with age across the 158 sessions. (B) Male participants showed a larger amplitude of the BOLD–CSF coupling as compared with females (p = 0.032) after controlling for disease condition. (C) The age-, gender-, and disease condition- adjusted BOLD–CSF coupling amplitude gradually decreased as the APOE ε4 allele number increases, but this change is not statistically significant (p = 0.22). Error bar in this figure represents the SEM. The underlying data can be found in ((TIF)Click here for additional data file.S3 FigA, B) The BOLD–CSF coupling adjusted for age and gender is significantly correlated  with the gray matter Aβ SUVRs (A) but not the 2-year longitudinal Aβ changes (B) across sessions of the MCI and SMC groups.  The BOLD–CSF coupling adjusted for age and gender is significantly correlated with  the 2-year longitudinal changes of MMSE scores (D) but not the baseline values (C) across sessions from MCI and SMC. Each dot represents a session. MCI and SMC sessions are colored with light gray and dark gray, respectively. The underlying data can be found in ((TIF)Click here for additional data file.S4 FigA, B) The BOLD–CSF coupling adjusted for age and gender is significantly correlated  with the gray matter SUVRs (A) but not the 2-year longitudinal SUVR changes (B) when the reference region is defined as the eroded white matter [(e matter . The res(TIF)Click here for additional data file.S5 FigA, B) BOLD–CSF coupling adjusted for age and gender is significantly correlated  with the gray matter Aβ SUVRs (A) but not the 2-year longitudinal Aβ changes (B) across participants. The composite reference region is the same as C, D) The association between the age- and gender-adjusted BOLD–CSF coupling and the 2-year longitudinal MMSE changes is significant  (D) but its correlation with the baseline MMSE score is not significant (p = 0.99) (C). The linear regression lines were estimated based on the linear least-squares fitting [( fitting . Each do(TIF)Click here for additional data file.S6 FigA, B) The BOLD–CSF coupling adjusted for age, gender, and mean FD is significantly correlated  with the cortical Aβ SUVRs at baseline (A) but not their changes in the following 2 years (B).  The association between the age-, gender-, and mean FD-adjusted BOLD–CSF coupling and the 2-year longitudinal MMSE changes is significant  (D) but its correlation with the baseline MMSE score is not significant (p = 0.85) (C). The linear regression lines were estimated based on the linear least-squares fitting. Each dot represents a single session. The underlying data can be found in ((TIF)Click here for additional data file.S7 Figp > 0.05, linear mix model adjustment was used). The linear regression lines were estimated based on the linear least-squares fitting. AD, MCI, SMC, and HC sessions are colored with blue, light gray, dark gray, and orange, respectively. The underlying data can be found in The mean FD was not significantly correlated with age, cortical Aβ SUVRs at baseline, 2-year MMSE change, or the BOLD–CSF coupling across sessions ((TIF)Click here for additional data file.S8 FigA–D) Similar to the main finding in Fig 3, the BOLD–CSF coupling is significantly correlated with cortical Aβ (A) and MMSE changes in the subsequent 2 years (D) across sessions in ADNI 2. (E–H) Very similar results  were found when we regressed the cohort information  and repeated the coupling–marker correlation analyses. The underlying data can be found in ((TIF)Click here for additional data file.S9 FigA) The cortical Aβ SUVRs and (B) the 2-year longitudinal MMS changes were correlated  with the BOLD–CSF correlations at different time lags after adjusting for age and gender. Black dots indicate significant correlations (p < 0.05). Red arrows indicate the lag (+3 seconds) that we used in the “BOLD–CSF coupling” . The underlying data can be found in ((TIF)Click here for additional data file.S10 FigA) The BOLD–CSF coupling adjusted for the cortical Aβ SUVR shows a significant correlation  with age across the 158 sessions. (B) Male participants showed a stronger BOLD–CSF coupling  as compared with females (p = 0.06). (C) The BOLD–CSF coupling, after adjusting age, gender, and cortical Aβ SUVR, gradually decrease from the HC, to SMC, to MCI, and then to AD group, but the change is not statistically significant (p = 0.32). (D) The age-, gender-, and cortical-Aβ SUVR adjusted BOLD–CSF coupling is not significantly (p = 0.86) correlated with the APOE ε4 allele. Error bar in this figure represents the SEM. The underlying data can be found in ((TIF)Click here for additional data file.S11 FigA) The association between the BOLD–CSF coupling (adjusted for age and gender) and the arousal index has a similar trend as Fig 4A.  The arousal index, adjusted for age and gender, is not significantly correlated with either the cortical Aβ level (B) or the 2-year longitudinal change of MMSE score (C).  The BOLD–CSF coupling remains to be significantly correlated with the cortical Aβ level (D) and the 2-year MMSE changes (E) after adjusting for age, gender, and arousal index. AD, MCI, SMC, and HC sessions are colored with blue, light gray, dark gray, and orange, respectively. Each dot represents a session. The underlying data can be found in ((TIF)Click here for additional data file.S12 FigA) The global BOLD signal and the CSF signal (both were represented with the percentage changes) from a representative participant showed corresponding changes (indicated by black arrows). (B) The BOLD–CSF cross-correlation functions from each of 158 sessions (gray curves). The black line represents the mean, whereas the error bars represent the SD across all the session. The underlying data can be found in ((TIF)Click here for additional data file.S1 Tablep-Values are derived from 2-sample t test for continuous measures and from Fisher exact test for categorical measures. The underlying data can be found in (DOCX)Click here for additional data file.S1 DataThis zipped folder contains Excel tables organized by figure and table, i.e.,  within w(ZIP)Click here for additional data file.


Arabidopsis SOC1, is a major positive regulatory factor of tomato inflorescence architecture by map-based cloning. High expression levels of STM3 underlie the highly inflorescence-branching phenotype in ST024. STM3 is expressed in both vegetative and reproductive meristematic tissues and in leaf primordia and leaves, indicative of its function in flowering time and inflorescence-branching development. Transcriptome analysis shows that several floral development-related genes are affected by STM3 mutation. Among them, FRUITFULL1 (FUL1) is downregulated in stm3cr mutants, and its promoter is bound by STM3 by ChIP-qPCR analysis. EMSA and dual-luciferase reporter assays further confirmed that STM3 could directly bind the promoter region to activate FUL1 expression. Mutation of FUL1 could partially restore inflorescence-branching phenotypes caused by high STM3 expression in ST024. Our findings provide insights into the molecular and genetic mechanisms underlying inflorescence development in tomato.Selection for favorable inflorescence architecture to improve yield is one of the crucial targets in crop breeding. Different tomato varieties require distinct inflorescence-branching structures to enhance productivity. While a few important genes for tomato inflorescence-branching development have been identified, the regulatory mechanism underlying inflorescence branching is still unclear. Here, we confirmed that SISTER OF TM3 (STM3), a homolog of  Oryza sativa), maize (Zea mays), and tomato (Solanum lycopersicum)1. Favorable inflorescence branching is always a major breeding target for achieving desirable production by balancing the sink-source relationship. Distinguished from the raceme-type inflorescences of Arabidopsis and panicle-type inflorescences of rice, tomato has a cyme-type inflorescence lacking a main axis, which initiates from a sympodial inflorescence meristem (SIM). The SIM generates a new SIM before terminating in a floral meristem (FM) and reiteration of this pattern produces tomato inflorescences and determination of branching4.Higher plants exhibit various inflorescence architectures progressing in complexity from a solitary flower to complex structures that contain multiple branches and flowers. The architecture of inflorescences is one of the determinant traits for many crops, such as rice , and FASCIATED INFLORESCENCE (FIN), cause meristems to enlarge, leading to an increase in inflorescence branching in tomato13.A series of regulatory genes that have received much attention makes major contributions to inflorescence architecture in tomato by changing the inflorescence-branching pattern. A larger vegetative shoot apical meristem (SAM) often produces more branching inflorescences. The WUSCHEL-CLAVATA (WUS-CLV) feedback regulatory loop is conserved maintaining the balance among SAM activities and controlling meristem size in single-flower truss (sft), is late-flowering and disrupts normal tomato sympodial growth, which reverses the inflorescence toward vegetative functioning after the initiation of one or a few flowers depending on the growing conditions16. The jointless (j) mutant produces indeterminate inflorescences that revert to vegetative growth after the production of two or three flowers17. Moreover, J and SFT cooperatively regulate the architecture of inflorescences preventing early changes in inflorescence meristem (IM) identity once inflorescence morphogenesis is initiated18. In addition, FALSIFLORA (FA), the tomato ortholog LEAFY, controls flowering time and floral meristem identity. The FA mutation results in the conversion of flowers into secondary shoots and produces a highly branched inflorescence20. In addition to these promotion-flowering genes, TERMINATING FLOWER (TMF) encodes an ALOG family protein and affects inflorescence organization in tomato. The tmf mutants flower early and convert multiflowered inflorescence into a solitary flower21. TMF and SlBOPs act synergistically to prevent precocious flowering and promote inflorescence complexity. All inflorescences on the slbop1/2/3 triple mutant develop only one or two flowers22.During phase transitions, flowering time genes are also important players in regulating inflorescence architecture in tomato. The florigen mutant, ANANTHA (AN) and COMPOUND INFLORESCENCE (S) delay the progression of an IM to the floral meristem (FM), resulting in additional branching23. JOINTLESS2 (J2), ENHANCER-OF-JOINTLESS2 (EJ2) and LONG INFLORESCENCE (LIN), three SEPALLATA 4 (SEP4) proteins, have redundant roles in inflorescence branching and cause a quantitative range of inflorescence branching with combinations of homozygous and heterozygous J2 and EJ2 mutations in tomato26. However, reconfiguration of inflorescence branching for breeding higher yield varieties in tomato remains challenging due to the limited known loci or genes and the largely unknown underlying mechanisms.In addition, floral meristem identity genes have a profound influence on tomato inflorescence architecture. Loss of Arabidopsis29. In addition, SOC1 and three MADS-box transcription factors, SHORT VEGETATIVE PHASE (SVP), AGAMOUS-LIKE 24 (AGL24), and SEP4, act redundantly and directly suppress TERMINAL FLOWER 1 (TFL1) to control Arabidopsis inflorescence architecture30. STM3, a homolog of SOC1 in tomato, also regulates inflorescence branching26. However, the mechanism by which STM3 positively regulates inflorescence branching in tomato is still unknown. In our study, we confirmed that STM3 is a major positive factor controlling inflorescence branching in tomato. Mutation of STM3 reduced the number of inflorescence branches, and overexpression of STM3 produced compound inflorescences. STM3 directly binds the promoter of FRUITFULL1 (FUL1) and activates its expression in vitro and in vivo. Mutation of FUL1 could partially restore inflorescence-branching phenotypes caused by high STM3 expression. Our findings provide insights into the molecular and genetic mechanisms underlying inflorescence development in tomato.SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) was first identified as an important flowering time integrator that regulates the floral transition in j2 and ej2 mutants31, from a set of 201 stable recombinant inbred lines (RILs) generated from a cross of Solanum Lycopersicum var. cerasiforme LA1310 (CC) and S. Lycopersicum Moneymaker (MM)32. The parental CC was generally a simple inflorescence, and less than 20% of inflorescences had two branches. Compared with CC, MM showed complex inflorescence branching, and more than 60% of inflorescences had more than two branches , on chromosome 1 was identified  by marker screening. We compared inflorescence-branching number among NIL-mib1MM, NIL-MIB1CC, and NIL-MIB1CM and found that NIL-mib1MM plants had significantly more branches than NIL-MIB1CC plants, and the heterozygous NIL-MIB1CM line showed an intermediate extent of inflorescence branching that is consistent with F1 hybrids  and Solyc01g093965 (TM3)  and found that the expression levels of both STM3 and TM3 were higher in ST024 than in ST147. Higher expression levels were also detected in NIL-mib1MM than in NIL-MIB1CC  Fig. . We obta/CC Fig. , suggest1CC Fig. , c. ThesSTM3 and TM3 led to more inflorescence branching in ST024, we transformed ST147 with vectors carrying the full-length CDS of TM3 and STM3 fused with YFP-HA or GFP-FLAG tags, respectively, and driven by the 35S promoter. Two independent overexpressed transgenic lines for each construct were obtained  leaves, in which STM3-FLAG was transiently expressed together with YFP-HA, STM3-YFP-HA, and TM3-YFP-HA, confirmed that STM3 could be immunoprecipitated by STM3-YFP-HA and TM3-YFP-HA fusion proteins but not by the YFP-HA control . The principal component analysis (PCA) results showed that the sequencing data were highly reproducible  were identified in tm3cr-1, stm3cr-1, and stm3 tm3cr-1, respectively , APETALA1/AP1 (Solyc02g089210MACROCALYX/MC (Solyc05g056620), SEPALLATA2/SEP2 (Solyc02g089200), and SEPALLATA3/SEP3 (Solyc05g015750), whose Arabidopsis homologs are related to floral meristem establishment, showed reduced expression levels in the single and double mutants35  were identified on the FUL1 promoter by JASPAR40  using a 49 bp DNA fragment of the FUL1 promoter containing S1 and the recombinant protein of STM3 with an MBP tag at the N-terminus (MBP-STM3). We found that MBP-STM3 but not MBP bound the FUL1 probe. In addition, the excess unlabeled oligonucleotide probe can compete with the labeled probe, and with the increased content of the competed unlabeled oligonucleotide probe, fewer labeled probes are bound by STM3. Moreover, when the CArG box motif of the probe was mutated, the binding of STM3 was obviously reduced  and S. Lycopersicum var. cerasiforme LA1310 (CC) were used as materials for the assessment of inflorescence branches as we described previously32. The NILs were generated by crossing ST147 and ST024. Then, heterozygous offspring at the qMIB1 locus were selected for selfing. After six generations, we used markers to select plants homozygous for MIB1CC or mib1MM and to determine the introgressed region (~191 kb) around the qMIB1 locus. Seedlings of RILs, NILs, and transgenic plants were grown in a commercial nursery for 30–40 days and then transplanted to fields. For inflorescence-branching assessment, all tomato plants were grown in a greenhouse at the Shunyi experimental station in Beijing, China. At least six individual plants of each accession were used for inflorescence-branching examination. From each plant, 5−6 inflorescences were evaluated. The % of total inflorescences represents the proportion of the number of inflorescences within a certain range to a total number of inflorescences.RILs derived from 2 population derived from the cross between two RILs, ST147 and ST024, with the j2delej2W genotype was used for mapping. We selected 24 plants with extremely compound inflorescences (mean number of inflorescence branches >200) and 31 plants with simple inflorescences (mean number of inflorescence branches <1.5) for bulked segregant analysis (BSA). Equal amounts of tissue from each plant were pooled for DNA extraction using standard protocols. The genomic DNA was sheared using a Diagenode Bioruptor Plus instrument to obtain ~300 bp fragments. Libraries were prepared using the NEXTflexTM Rapid DNA-Seq Kit for Illumina (NOVA-5144-08) according to the manufacturer’s protocol. Genomic DNA reads were trimmed by quality using Trimmomatic47, and paired reads were mapped to the reference tomato genome (SL2.50) using BWA-MEM48. SNP calling was performed as described previously49. BSA was performed with modification50. SNPs between two parental genomes were identified for further analysis when the base quality value was ≥20 and the SNP quality value was ≥20. On the basis of these criteria and the number of SNPs with a read depth ≥5, an SNP index was calculated for both bulk samples expressing the proportion of reads harboring SNPs that were identical to those in the parent (ST147). The ∆SNP index was obtained by subtracting the SNP index for the simple inflorescence bulk sample from that for the compound inflorescence bulk sample. An average SNP index for the compound and simple inflorescence bulk samples was calculated using a 1,000 kb sliding window with a step size of 10 kb. For fine mapping qMIB1, 38 recombinant plants were selected according to the region determined by BSA-seq from 960 F2 seedlings and self-pollinated in the greenhouse. The progeny of ten recombinant plants was selected, and their inflorescence-branching number was observed.An FUBI3 (Solyc01g056840) was used as the internal control for qRT-PCR. The primers used for qRT-PCR are listed in Supplementary Table RNA was isolated using TRIzol Universal reagent . DNA contamination was removed using the TURBO DNA-free Kit . Reverse transcription was performed with 5×All-In-One MasterMix with AccuRT Genomic DNA Removal Kit  using 2 μg of total RNA. Quantitative PCR (qPCR) was performed with EvaGreen 2×qPCR MasterMix-No Dye  on a Bio-Rad CFX-96 Real-Time PCR instrument using the following program: 3 min at 95 °C followed by 40 cycles of 20 s at 95 °C, 30 s at 60 °C, and 20 s at 72 °C. tm3cr-1, stm3cr-1, and tm3 stm3cr-1. Three biological replicates were performed, and each biological replicate contained at least 200 individuals. A total of twelve RNA-Seq libraries were constructed and sequenced using Illumina HiSeq2000 at Berry Genomics (http://www.berrygenomics.com/). The filtered clean reads were aligned to the tomato genome (ITAG4.0) by STAR v2.5.3, and their features were counted by feature Counts v 1.5.3, as described in a previous paper51. The statistical package DEGseq with the MA-plot-based method in R version 3.0.3 was used to calculate the P value, which was adjusted using the Benjamini-Hochberg procedure. The fold change between the ST024 and tm3cr-1, ST024 and stm3cr-1, and ST024 and stm3 tm3cr-1 libraries was calculated as FPKM (fragments per kilobase of transcript sequence per million base pairs sequenced). The thresholds for the identification of DEGs were as follows: FPKM > 1 in any tissue, fold change > 1.5 or < 0.6666, and Benjamini-Hochberg adjusted P value < 0.05. GO analyses of DEGs were performed using their best homologous genes in Arabidopsis with DAVID .Total RNA was isolated from reproductive meristematic tissues collected from 21-d-old seedlings of ST024, A SMARTer RACE cDNA Amplification Kit  was used for the RACE assay according to the manufacturer’s instructions. Approximately 1 μg of total RNA was used to prepare RACE-Ready first-strand cDNA. Then, the 3′ and 5′ ends of cDNA were amplified by using the 3′ or 5′ gene-specific primers listed in Supplementary Table http://cbi.hzau.edu.cn/CRISPR2/). Primers containing sgRNAs and BsaI recognition sites were used to amplify the sgRNAX_U6-26t_SlU6p_sgRNAX fragments using a pCBC_DT1T2_SlU6p vector as the template, after which the fragments were purified and cloned into pTX041 at the BsaI sites53. To generate 35S::STM3-YFP-HA, 35S::TM3-YFP-HA, and 35S::STM3-GFP-FLAG constructs, the coding sequences of STM3 and TM3 were fused to the N-terminus of YFP-HA or GFP-FLAG, respectively. All plasmids were validated by sequencing and then transformed into the Agrobacterium tumefaciens strain AGL1. The transgenic plants were validated by PCR and sequencing. All primers used are listed in Supplementary Table For CRISPR/Cas9 constructs, two sgRNA binding sites per gene were selected with the CRISPR-P v2.0 tool  paraformaldehyde buffered with phosphate-buffered saline . Fixed tissues were dehydrated in a graded ethanol:histochoice  series and impregnated with paraplast . Dewaxed thin sections (10 μm) were hybridized with hydrolysis probes for 12 h at 55 °C. Complete color development sections were observed using a fluorescence microscope .alpha-Gal.Yeast two-hybrid assays were performed according to the Yeastmaker™ Yeast Transformation System 2 user manual . To explore interactions between STM3, TM3, and itself, the full-length coding sequence of each gene was cloned into bait and prey vectors, pGBKT7 and pGADT7, respectively. A pair of bait and prey plasmids were cotransformed into the Y2H gold yeast strain according to the Clontech yeast protocol handbook instructions. The resultant strains were subsequently grown on plates lacking leucine and tryptophan for 3 days at 30 °C. The interaction was tested via growth assays on media lacking leucine, tryptophan, histidine, and adenine but containing X-55. The coding sequences of STM3 and TM3 were fused to the N-terminus of FLAG or YFP-HA tags to construct 35S::STM3-FLAG, 35S::STM3-YFP-HA, and 35S::TM3-YFP-HA vectors. These plasmids were transformed into A. tumefaciens strain EHA105 and then coinfiltrated into the leaves of 4-w-old N. benthamiana plants together with P19. Equal numbers of samples were collected two days after infiltration, ground in liquid nitrogen, and then homogenized in 1 ml of extraction buffer: 50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.19% CA630; 20% glycerol; 5 mM DTT; and 1 tablet/50 ml of protease inhibitor cocktail . The lysates were mixed sufficiently and then centrifuged at 16,000 × g for 10 min at 4 °C. In addition, 5 μg anti-HA  antibody and 20 μl Dynabeads Protein G  were incubated for 2 h at 4 °C. The lysates and the HA-binding beads were incubated overnight at 4 °C. The incubated beads were washed five times with 1 × PBS. Then, 30 μl of 6× protein loading buffer was added and boiled for 5 min. The proteins were electrophoretically separated by 10% SDS–PAGE and transferred to a PVDF membrane . Immunoblots were performed using an anti-FLAG antibody  for STM3-FLAG. The bands were visualized on a Tanon-5200 Chemiluminescent Imaging System (Tanon Science and Technology).The Co-IP assay was performed as previously described with minor modifications35S::STM3-YFP-HA transgenic plants as described previously with minor modifications32. Meristems were completely ground in liquid nitrogen and cross-linked in 1% formaldehyde (Sigma-Aldrich) for 10 min at 4 °C. Chromatin was sheared using a Diagenode Bioruptor Plus instrument to obtain ~300 bp DNA fragments. Anti-HA  was used for immunoprecipitation. The DNA isolated by ChIP was used for qPCR analysis or Illumina paired-end sequencing. qPCR was performed using EvaGreen 2×qPCR MasterMix-No Dye  on a Bio-Rad CFX-96 Real-Time PCR instrument with the following program: 3 min at 95 °C followed by 50 cycles of 20 s at 95 °C, 30 s at 60 °C, and 20 s at 72 °C. The intergenic region around ACTIN (Solyc03g078400) was used as an internal control. Primers for qPCR are listed in Supplementary Table ChIP was performed using 0.5 g hand-dissected meristems of 18-d-old seedlings of STM3 were amplified by PCR using the primer pair in Supplementary Table 56. All recombinant proteins were expressed and purified from Escherichia coli strain BL21 RIL . The MBP-STM3 protein and MBP protein were induced by 0.2 mM isopropylthio-b-D-galactoside at 16 °C for 16 h. Ni-NTA agarose  was used to purify MBP-STM3 and MBP according to the manufacturer’s instructions. We synthesized and annealed the 5′ biotin DNA probes used in EMSA. DNA gel shift assays were performed using the LightShift Chemiluminescent EMSA kit . Each EMSA binding reaction (20 μL) contained 0.5 μL purified recombinant protein, 4 μL biotin-labeled probe DNA (100 pmol), 2 μL binding buffer, and 1 μL poly (dI-dC). Transferred DNA and protein were cross-linked using a UV lamp at 312 nm. The biotin-labeled DNA was determined using a Thermo Scientific chemiluminescence kit. The bands were visualized on a Tanon-5200 Chemiluminescent Imaging System (Tanon Science and Technology).The full-length coding regions of 57. The nearly 2.3 kb FUL1 promoter sequence was amplified using MM genomic DNA as a template and integrated into pluc-35Rluc using the in-fusion HD cloning kit . Then, the mutated FUL1 promoter was generated by deleting the 40 bp sequence including the CArG box in the S1 region of the promoter  medium until the OD600 value reached 1. The A. tumefaciens cells were collected by centrifugation and suspended using 10 mM MgCl2 and 150 μM acetosyringone. The cells containing the overexpression plasmids, luciferase plasmid, and p19 plasmid were mixed in a volume ratio of 2:1:3 and infiltrated into Nicotiana benthamiana leaves using a syringe. The leaves were harvested and ground in liquid nitrogen at 2 d after infiltration. The activities of firefly luciferase and Renilla luciferase were measured using a dual-luciferase reporter assay system  on a Promega GLOMAX 20/20 LUMINOMETER. Thirty-seven sets of data were collected from the different leaves. The measured data (LUC/REN) were normalized to the control on the same leaf.For plasmid construction, the pluc-35Rluc backbone vector was obtained from pPZP211ter Fig. . The prisupplementary table S1all supplementary figuresSupplementary Table S2Supplementary Table S3Supplementary Table S4


The myodural bridge (MDB) connects the suboccipital musculature to the spinal dura mater (SDM) as it passed through the posterior atlanto-occipital and the atlanto-axial interspaces. Although the actual function of the MDB is not understood at this time, it has recently been proposed that head movement may assist in powering the movement of cerebrospinal fluid (CSF) via muscular tension transmitted to the SDM via the MDB. But there is little information about it. The present study utilized dogs as the experimental model to explore the MDB’s effects on the CSF pressure (CSFP) during stimulated contractions of the suboccipital muscles as well as during manipulated movements of the atlanto-occiptal and atlanto-axial joints. The morphology of MDB was investigated by gross anatomic dissection and by histological observation utilizing both light microscopy and scanning electron microscopy. Additionally biomechanical tensile strength tests were conducted. Functionally, the CSFP was analyzed during passive head movements and electrical stimulation of the suboccipital muscles, respectively. The MDB was observed passing through both the dorsal atlanto-occipital and the atlanto-axial interspaces of the canine and consisted of collagenous fibers. The tensile strength of the collagenous fibers passing through the dorsal atlanto-occipital and atlanto-axial interspaces were 0.16 ± 0.04 MPa and 0.82 ± 0.57 MPa, respectively. Passive head movement, including lateral flexion, rotation, as well as flexion–extension, all significantly increased CSFP. Furthermore, the CSFP was significantly raised from 12.41 ± 4.58 to 13.45 ± 5.16 mmHg when the obliques capitis inferior (OCI) muscles of the examined specimens were electrically stimulated. This stimulatory effect was completely eliminated by severing the myodural bridge attachments to the OCI muscle. Head movements appeared to be an important factor affecting CSF pressure, with the MDB of the suboccipital muscles playing a key role this process. The present study provides direct evidence to support the hypothesis that the MDB may be a previously unappreciated significant power source (pump) for CSF circulation. Recently, several published studies have identified the existence of the MDB in mammals 6, birds8 and reptiles9, that the MDB is an evolutionary conserved physical structure that is involved in important physiological functions.The MDB is a complex anatomical structure that physically connects the cervical spinal dura mater (SDM) to the suboccipital muscles, including the rectus capitis posterior minor (RCPmi), the rectus capitis posterior major (RCPma), and the obliquus capitis inferior (OCI) while passing through the posterior atlanto-axial and atlanto-occipital interspaces1. Moreover, that the suboccipital muscles, along with MDB might play an essential role in transmitting cervical proprioception by monitoring tensional changes in the cervical SDM and effecting a rapid adjustment of the head position10. Furthermore, as the MDB might prevent the cervical SDM from folding inward the spinal cord during head and neck movements thus preventing the impedance of cerebrospinal fluid (CSF) flow in the subarachnoid space and cisterna magna11. Other recent studies have postulated that during head movement, the MDB would pull on the cervical SDM producing changes in the volume and pressure of the CSF located in the cisterna magna and subarachnoid space of the upper cervical spine, consequently promoting the circulation of the cerebrospinal fluid at the occipito-cervical junction14. A recent study using the PC cine of MRI evidenced a significant increase in the net flow of cerebrospinal fluid in the cephalic direction after head rotation15, providing indirect evidence for the hypothesis that the MDB serves as one of the power sources for CSF circulation.Concerning the functionality of the MDB, it has recently been proposed that contraction of the suboccipital muscles, produced during head movement, might assist in resisting SDM in-folding20. However, its mechanism is still unknown up to now. The basic research on the function of the myodural bridge is very important for the study of the mechanism of clinical disease.Clinically, dysfunctional suboccipital musculature was observed to be related to various types of headachesTo test the proposed “cerebrospinal fluid circulation dynamic hypothesis” relating to the myodural bridge, the present study explored the effects of suboccipital muscles contractions and head movements on the CSF pressure and the potential role of MDB.Seventy-three healthy adult Beagle dogs  were provided after experimental teaching of surgery for medical students by the Experimental Animal Center of Dalian Medical University. All animal experiments conformed to ethical requirements of the school.Ten Beagle dogs were euthanized. Afterwards, the nuchal region of each dog was dissected, layer by layer, to explore the subocciptal musculature.The rectus capitis dorsal minor (RCDmi) muscle was fully exposed. Fibrous connections between the RCDmi and the dorsal atlanto-occipital membrane (DAOM) were observed as well as fibrous connections between the DAOM and the upper cervical SDM.The short head of the rectus capitis dorsal major (RCDma) and the obliquus capitis inferior (OCI) muscles were exposed integrally. Fibrous connections between these two muscles and the dorsal atlanto-axial membrane (DAAM) were observed as well fibrous connections between the DAAM and the upper cervical SDM.Utilizing six of the euthanized Beagle dogs, tissue-blocks from the sub occipital region were prepared, which included the suboccipital muscles, the cervical dura mater, and tissues between them at the level of the dorsal atlanto-occipital interspace or dorsal atlanto-axial interspace. These tissue blocks were conventionally sectioned and stained with Masson trichrome and HE. The fibrous connections among the suboccipital muscles, DAOM (or DAAM) and SDM were observed and photographed under the light microscope.Tissue blocks taken from eight of the euthanized Beagle dogs at the level of the dorsal atlanto-occipital interspace, consisted of the RCDmi, the DAOM and the SDM were dissected out.At the level of the dorsal atlanto-axial interspace, tissue blocks consisting of the OCI, the short head of the RCDma, the DAAM, and the SDM were obtained.These specimens all underwent conventional preparation for SEM evaluations. The fibrous connections between the DAOM (or DAOM) and the SDM at the dorsal atlanto-occipital and atlanto-axial interspaces were observed and photographed under the SEM.Tissue blocks were taken from the euthanized the Beagle dogs N = 13) were utilized for this experiment. In the dorsal atlanto-occipital interspace, the specimens were intact tissue blocks containing the RCDmi, the DAOM, the SDM, and the fibrous connections  was then dropped into the narrow gap between the SDM and the other tissues to evidence the contour of the connected area between the MDB and the SDM  × 64) software.The SDM, the DAOM (or DAAM), and the muscles (RCDmi and OCI) were placed on a plastic film while maintaining their original SDM Fig. . After sThe fresh specimens were sealed in airtight bags and stored at 4 degree (C). Before measurement the specimens were left at room temperature for half an hour. Uniaxial tension experiments were performed at room temperature within the post mortem time of 10hrs. The specimen was fixed on the test rig as shown in Fig. Pre-operation preparation: 33 animals were used for this aspect of the study. 3% Nembutal was injected into the animal intraperitoneally for anesthesia with tracheal intubation.Cerebrospinal fluid pressure monitoring: The puncture needle was inserted into the subarachnoid space through the dorsal space between L4 and L5. When cerebrospinal fluid (CSF) appeared at the opening of the puncture needle, a catheter was inserted 24 cm cranially into the spinal canal and then connected to a blood pressure sensor  = 0.006 mmHg) to record the CSF pressure by BL-420F biological data acquisition and analysis system  during the experiment.Blood pressure monitoring: An arterial intubation was performed into the femoral artery and was monitored with a blood pressure sensor (PT-102) to record the blood pressure and the heart rate by the BL-420F system.Respiration monitoring: A bandage type respiratory sensor (HX200) was fixed to the chest of the dogs, and the frequency and amplitude of their respiration were recorded with the BL-420F system.Exposure of suboccipital muscles: The superficial structures were reflected layer by layer until the suboccipital muscles were exposed.The BL-420F biological function laboratory system was used for this aspect of the study. Electrical stimulation was set at an output voltage of 10 V, wavelength of 30 ms and 30 Hz, and the electrical stimulation was performed thrice for 10 s in each side of the long and short heads of the RCDma and the OCI muscles, respectively. The CSF pressure, respiration, and blood pressure were all recorded simultaneously.The spinous process of the axis (C2) was fixed with a custom made cervical fixation device to stabilize the neck during passive movements of the head. Lateral flexion, flexion–extension and rotations of the head at the atlanto-occipital and atlanto-axial joints were respectively performed 10 times, with the frequency of motion being once per second. The CSF pressure, respiration,and blood pressures were monitored and recorded.The stimulating electrode was inserted into the left and right side of the RCDmi respectively, and then electrical stimulations were given with a stimulus intensity of 10 V for 10 s, thrice for each side. The CSF pressure, respiration, and blood pressure were observed and recorded simultaneously.In 6 animals, the left OCI muscle was disconnected from its deep structures and the right side was not treated as a control group. Subsequently the OCI was electrically stimulated on the left and right side respectively, as the CSF pressure, respiration, and blood pressures were monitored and recorded.The experimental dogs were euthanized by air embolism under anesthesia .The experimental data was recorded before and after the electrical stimulations by using the Biological function laboratory system machine, and peak value of pressure wave was collected. The data were recorded as: Mean ± SD and analyzed by paired samples t-test and rank sum test (Wilcoxon signed-rank), P < 0.05 was chosen to be a statistically significant result.All animal experiments conformed to ethical requirements of the school.The experiment was performed under anesthesia, which may be different from the physiological condition; Because of the great difficulty of surgery, the number of cases of myodural bridge disconnection was relatively small, so statistical analysis cannot be realized.All of the described experiments were approved by the Animal Experimental Center of Dalian Medical University.The present study was designed, performed, and reported according to the principles of the ARRIVE guidelines.All methods were carried out in accordance with relevant guidelines and regulations. All experimental protocolswere approved by Dalian medical university.In the suboccipital region, three suboccipital muscles were identified, the RCDma and RCDmi, and OCI. The RCDma was observed to have long and short heads Fig. a,f.FigurThe RCDmi was observed to cross over the dorsal atlanto-occipital interspace Fig. a, givingThe short head of the RCDma as well as the OCI were both observed to pass over the dorsal atlanto-axial interspace. The short head of the RCDma was found to cover the small central part of this space and the OCI covered the large lateral part of this space Fig. f. Deep tAt the level of the dorsal atlanto-occipital interspace, it was found that abundant dense collagen fibers from the ventral side of the RCDmi were inserted into the DAOM. The DAOM subsequently gave off collagen fibrous clusters from its ventral side and connected to the SDM Fig. a-d.At the dorsal atlanto-axial interspace, the cranial part of the DAAM was composed of abundant concentrated dense connective fibers, whereas the caudal part of the DAAM evidenced loose fibers, having scattered parallel running collagen fibrous bundles Fig. e-g.Dorsal to the DAAM, numerous fibrous collagen bundles were found originating from the ventral aspect of the OCI and dorsal end of the short head of the RCDma and inserted into the cranial dense part of the DAAM, and continuously the DAAM gave off abundant collagen fibers and then fused with the SDM. Fig. e-g.FigurFor convenience of observation, the DAOM and SDM were separated a little apart and then lots of cord-like structures were found connecting them. These plexiform cords were originated from the ventral side of the DAOM and then fused together with the SDM Fig. a-c. Betw2. The fracture force of the MDB was 7.55 ± 1.74 N, and the ultimate tensile strength amounts to 0.16 ± 0.04 MPa. The RCDma and OCI’s MDB was insert into the SDM covering 18.60 ± 11.62 mm2. The fracture force of the MDB was 13.80 ± 10.43 N, and the ultimate tensile strength 0.82 ± 0.57 MPa.The RCDmi’s MDB was inserted into the SDM covering about 47.58 ± 10.80 mmThe results showed that the CSF pressure of the specimens were significantly increased from 12.41 ± 4.58 mmHg to 13.45 ± 5.16 mmHg during electrical stimulation of the OCI  was also found to participate the MDB at the posterior atlanto-axial and atlanto-occipital interspaces respectively29. Up to now, the MDB has been proved to be a constant anatomical structure in the occipito-cervical junction. But its function is still unclear. Recently, a new functional hypothesis was proposed that the MDB complex might be one of the sources of power for CSF circulation22.In the recent 30 years, the MDBs have been discovered constantly between the suboccipital muscles and the upper cervical spinal dura mater (SDM) in the occipitocervical junction in human. The MDB of RCPmi is located in the posterior atlanto-occipital interspace30 suggested that the MDB might be a common existence and a normal anatomical structure in mammals. The present animal study provides further evidence for the ubiquity of MDB in mammals.In this study, combining the multi-methods of gross dissection, histology, and scanning electoral microscope, it was provided that the MDB was compose of abundant dense connective fibers existing between the dorsal occipital muscles  and the SDM. Recently, Zheng et al.31. Although the MDBs are different from the arterioles in structure, the UTS of the MDB is similar to that of the arteriole. This allows us easily to perceive the strength of the dural bridge by comparing the MDB with the arterioles in the epidural space.Based on the MDB structure, the tensile strength of the MDB was managed to be measured. The rupture of the myodural bridge complex (MDBc) occurred at site between the DAOM (or DAAM) and the SDM, which may be the weakest part of the MDBc. The ultimate tensile strength (UTS) of the MDB at the dorsal atlanto-occipital and atlanto-axial interspaces are 0.16 ± 0.04 MPa and 0.82 ± 0.57 MPa respectively, and the latter is obviously stronger than the former. It has been reported that the UTS of the arterial wall is 0.5–1.72 MPa32, with one order of magnitude stronger than that of the MDB, and this may accord with the primary and secondary relationship between structures and is also necessary to ensure the structural safety of the dura mater. In addition, tendons and ligaments are tough soft tissue structures33, and the UTS of the MDB attached to the dura mater is 40 to 2000 times weaker than tendons or ligaments. However, their physiological functions are also significantly different.The dural is a membranous structure, with cerebrospinal fluid in the dural sac, venous plexus and loose connective tissue in the epidural space. The resistance of the dura movement would be small, and the UTS of the MDB attached to the dura mater may be enough to complete the bridge function of pulling the dura mater. And the strength of the dura mater is within the range of 3.28–7.86 MPaThis study defines the mechanical properties of the MDB and provides data and method for further investigation on the biomechanical characteristics of the MDB.In this study, we demonstrated the effect of stimulated contractions of the suboccipital muscles and passive movements of the atlanto-occiptal and atlanto-axial joints on the CSF pressure. The results showed that the electrical stimulation of the OCI caused a significant increase in the CSF pressure, while this effect was eliminated by cutting the myodural bridge of OCI in advance. Also in vivo remaining dorsal occipital muscles intact, there was an obvious rise in the CSF pressure during passive head rotations. Head rotation mainly occurs at the atlanto-axial joint, where the OCI muscle is involved. Combining anatomical and physiological results, it was proved that, during head rotation, the contraction of the OCI is probably transferred to the SDM via the MDB, and repeated traction on the spinal dural during head rotations might lead to reciprocating changes of geometrical shape of the upper cervical spinal dura capsule and then result in the subarachnoid space pressure increase with some kind of dynamic mechanism.Moreover, the present study found that passive head movements of lateral flexion and flexion–extension all made significant increase of the CSF pressure. During these kinds of head motion, one or more dorsal occipital muscles were involved at the occipito-atlantal joint, and the short head of RCDma and the RCDmi might pull the spinal dura capsule via their MDBs, and then cause a rise in the CSF pressure. Nevertheless, there was no obvious change in the CSF pressure when active electrical stimulation of the short head of RCDma or the RCDmi was performed. The RCDmi and the short head of RCDma are small muscles, and they could not move the the occipito-atlantal joint alone. So during their active electrical stimulation, they produced only isometric contractions and thus a negligible pull on the SDM was present without effect on the CSF pressure.20. The role of the suboccipital muscles via the MDB in CSF circulation might be its possible mechanism.Generally, the present results provide direct evidence that the MDB complex is one of the power sources for CSF circulation. Additional in clinic, it also sheds light on the possible relationship between pathologic suboccipital muscles and chronic headache. Clinical studies have shown that the dysfunction of the suboccipital muscles under various kinds of pathologic status might be correlated with chronic cervical headacheThe myodural bridge exists in the suboccipital region of dogs and has a tensile strength similar to the walls of small artery. The head movements are an important factor affecting CSF pressure, and the MDB of the suboccipital muscles may play a key role this process. The present study provides direct evidence to support the hypothesis that the MDB may be an important power source for CSF circulation.


Escherichia coli, Klebsiella pneumoniae, Thermothelomyces thermophila, Lactobacillus acidophilus, and Staphylococcus haemolyticus. To further explore their potential role in patients’ conditions, we examined their correlation with clinical parameters. Importantly, microbiome signatures with compositional changes were correlated with the C-reactive protein (CRP) level in blood and granulocyte percentage in CSF. Moreover, the blood in subsets of patients with a predominance of Klebsiella pneumoniae could replace CSF as the main specimen for clinical monitoring.Bacterial meningitis shows a higher incidence in children than adults, but signs may be scarce. Although some pathogenic microorganisms of meningitis from cerebrospinal fluid (CSF) have been reported, the signature of the representative microbiota in CSF and blood samples from patients remains incompletely revealed. To extend the understanding of the microbiome in patients, we recruited 32 children with bacterial meningitis, 30 undiagnosed infectious children, and 10 matched healthy individuals, which was followed by untargeted metagenomic next-generation sequencing (mNGS) and bioinformatic analysis. Our results showed that children with bacterial meningitis exhibited different microbiome signatures in their CSF and blood compared with undiagnosed and healthy children, and patients could be divided into varied subsets according to these signatures, including IMPORTANCE This study revealed the microbial compositions in children with bacterial meningitis who were treated with antibiotics and made a comprehensive comparison between blood and CSF specimens for the risk and prognosis assessment. We found that microbiome signatures could distinguish patient subsets in the children and were correlated with the CRP level in blood and granulocyte percentage in CSF. The compositional changes in representative microbiota constituents could provide guidance for clinical monitoring and antibiotic intervention. Haemophilus influenzae, and Streptococcus pneumoniae, the mortality of this disease has been significantly reduced, but it is still between 5% and 15%  is a damaging infectious disease of the central nervous system in children, especially infants , and its and 15% , 4.Early and accurate diagnosis of BM is vital for implementing an effective therapeutic schedule, such as antibiotic treatment. However, it is often impossible due to the limitations of traditional approaches in terms of sensitivity and the time requirements of culture and bacterial cultivation. The current standard for diagnosing infections depends on differential patient history, clinical manifestations, and imaging detection, followed by laboratory testing. This conventional method is particularly challenging for meningitis due to the lack of diagnostic tests for uncommon pathogenic microorganisms and the limited availability and volume of cerebrospinal fluid (CSF) samples because of the invasiveness of the procedures, such as lumbar puncture. Hence, the cause of meningitis remains unexplained in a large proportion of patients .Metagenomic next-generation sequencing (mNGS) has a comprehensive performance in verifying nearly all probable microorganisms, including bacteria, viruses, fungi, and parasites, in a single test –9. Howev–10–Escherichia coli, Klebsiella pneumoniae, Thermothelomyces thermophila, Lactobacillus acidophilus, and Staphylococcus haemolyticus, and their composition patterns can divide patients into different subgroups. Their correlation with clinical parameters was assessed to further explore the potential role of representative microbiota constituents in patients’ conditions. The results showed that microbiome composition changes were associated with the C-reactive protein (CRP) levels in blood and granulocyte percentages in CSF. Furthermore, the subsets of patients with a predominance of Klebsiella pneumoniae could instead use blood for clinical monitoring. These results provide a significant reference for clinical monitoring and antibiotic treatment.We performed a prospective study involving hospitalized children diagnosed with BM. Recently, the sensitivity and specificity of mNGS of CSF have been proven in the identification of pathogens in the central nervous systems of infected patients , 14. OurWe enrolled 32 children with BM, 30 infected but undiagnosed individuals, and 10 healthy controls for this microbiome study. Notably, all children with BM received antibiotic treatment and were discharged upon improvement. A total of 102 DNA samples were extracted from specimens: 10 from healthy blood (NB), 31 from patient blood (PB), 31 from patient CSF (PC), and 30 from CSF from undiagnosed patients (IN) , we mainP = 5.3e−05 and P = 0.0078 for Shannon and Simpson indexes, respectively) between patient CSF (PC) and undiagnosed individuals’ CSF (IN)  of Bray-Curtis distance revealed that the microbiota of healthy controls (NB) and patients (PB) in blood formed two distinct clusters  and undiagnosed patients (IN)  displayed a significant difference (CSF (IN) . This reCSF (IN) . In all clusters . The larnts (IN) . Our fin10.1128/mSystems.00049-21.1FIG S1t test (t test), comparison of multiple groups using Kruskal-Wallis test. (B) Comparison of Shannon index of each group, comparison of two groups using Wilcoxon test, comparison of multiple groups using Kruskal-Wallis test. (C) Comparison of Simpson index of each group, comparison between two groups using Student’s t test (t test), comparison of multiple groups using Kruskal-Wallis test. (D) Comparison of Simpson index of each group, comparison between two groups using Wilcoxon test, comparison of multiple groups using Kruskal-Wallis test. Download FIG S1, TIF file, 1.4 MB.Diversity comparison among different groups. (A) Comparison of Shannon index of each group, comparison between two groups using Student’s Copyright © 2021 Liao et al.2021Liao et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Considering the different microbial compositions in blood and CSF, we wondered whether microbiota members can be used as biomarkers to differentiate NB and PB groups as well as PC and IN. To this end, we established a model using a random-forest machine learning method  to correEscherichia coli, Klebsiella pneumoniae, Thermothelomyces thermophila, Lactobacillus acidophilus, Staphylococcus haemolyticus, and Lactococcus lactis  (n = 6/32) (n = 47/62) (n = 12/62) . These results strongly indicated that the representative microbiota could serve as a signature for clinical stratification.To further explore the distribution of the target microbiome in BM children, we measured the relative abundance of a single bacterium (species counts/microbiota counts) in each patient. Interestingly, we found a specific combination pattern of the candidate microbiome. The number and species of bacteria among meningitis cases were obviously diverse , and patients with Klebsiella pneumoniae as the dominant pathogen  were assigned to PC_blue (n = 20), which was dominated by K. pneumoniae. Most patients with a predominance of E. coli (n = 13) in PB_red  were assigned to the PC_blue (n = 20) and PC_purple subgroups (n = 2); however, four cases in PB_red  were assigned to PC_green, whose primary species was K. pneumoniae. In short, the consistency of blood and CSF in patients (PB_green) with a predominance of K. pneumoniae was 100%, while the consistency was only 52.94% in the PB_red group. Consequently, these data show that the blood specimens of patients with a predominance of K. pneumoniae has the same detection power as their CSF specimens.Previous results have demonstrated that the dynamic distribution of target microbiota in specimens can reflect the infection and inflammation status of BM patients. We wondered whether blood has the same test effect as CSF; that is, can blood replace CSF in pathogen detection? Then, we analyzed the distribution consistency of the candidate microbiota in different patient subpopulations, taking blood subsets , particularly for energy production and conversion. Other functional categories, including amino acid transport and metabolism, nucleotide transport and metabolism, carbohydrate transport and metabolism, coenzyme transport and metabolism, lipid transport and metabolism, inorganic ion transport and metabolism, and secondary metabolites, were depleted in the PB group  isolated from neonatal E. coli meningitis  and transmigrate through HBMECs via an enclosed vacuole  specimen by lumbar puncture (LP) for analysis . However53–Escherichia coli gradually decreased, while the proportions of Klebsiella pneumoniae and Thermothelomyces thermophila increased, but not continuously. In the PC_purple, PC_red, PC_blue, and PC_green patient groups  accounted for only 35.47% of the candidate microbiota versus 64.53% for Escherichia coli, while in CSF, K. pneumoniae  accounted for 100%. Additionally, the recommended treatment for E. coli is ampicillin in combination with a broad-spectrum cephalosporin (cefotaxime or ceftazidime). These results indicate that the pertinence of empirical antibiotics needs to be improved, and the treatment strategy should be adjusted in a timely manner according to the dynamic changes in the microbiota. In addition, the incongruent response of blood and CSF to antibiotics may be attributed to the blocking effect of the BBB and the BCSFB. Therefore, the most appropriate drug regimen for patients with BM remains to be further discussed.According to Chinese neonatal BM treatment experience, meropenem (40 mg/kg of body weight) is generally used for Klebsiella pneumoniae had a consistent distribution pattern in blood and CSF; that is, patients with a high abundance of K. pneumoniae in blood would also have a high abundance of K. pneumoniae detected in CSF. In addition, the target microbiome affected the level of CRP in blood and the proportion of granulocytes (monocytes) in CSF, which further influenced the degree of infection and inflammation. Studies have shown that bacteria invade the body of children and enter intracranially through the BBB . The proliferation of pathogenic bacteria activates the host immune response, leading to the release of a large number of inflammatory factors, ultimately causing severe brain damage  to 10 healthy children based on blood data and compared 31 children with meningitis to 30 undiagnosed individuals based on CSF data. The criteria for eligibility in this study follow: (i) a diagnosis of bacterial meningitis, (ii) age from 0 to 14 years old, (iii) treatment with antibiotic(s), (iv) a discharged diagnosis of improvement, (v) collection of at least 350 μl blood, and (vi) collection of at least 350 μl CSF. All conventional detection of CSF was completed by the Laboratory Department of Beijing Children’s Hospital. Six patients had a positive pathogenic test result before sample collection, and 81.25% patients had a negative result. Thirty patients with undiagnosed infections were treated as positive controls, and 10 healthy children were treated as negative controls. The CSF and plasma samples of the first lumbar puncture of all meningitis patients were tested by metagenomic next-generation sequencing (mNGS) and traditional tests. All bacterial meningitis patients have received antibiotic intervention, and each child received an average of four antibiotics and 15 days of treatment. Among all antibiotics, cephalosporins, meropenem, vancomycin, amoxicillin, and fusidic acid were administered more frequently. Among them, cephalosporins mainly include cefaclor, cefdinir, cefepime, cefmenoxime, and cefoperazone-sulbactam. Due to the relatively high cost, each patient underwent only one mNGS examination.The study was approved by the Institutional Review Committee of the Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences, and Beijing Children’s Hospital. Written informed consent was obtained from all patients or their legal representatives, which complied with the guidelines of the Shanghai Institute of Nutrition and Health of the Chinese Academy of Sciences and the Beijing Children’s Hospital Institutional Review Committee.Three hundred fifty microliters of cerebrospinal fluid and plasma were aseptically collected and stored at −80°C. Metagenomic DNA was extracted immediately using Qiagen pathogen lysis tubes and QIAamp UCP pathogen minikit in the National Secondary Biosafety Laboratory (biosafety level 2 [BSL-2]) of the Shanghai Institute of Pasteur of the Chinese Academy of Sciences. Whole-genome amplification was performed using an amplification kit . All DNA libraries were constructed following the TruSeq protocol . Whole-genome amplification and library construction were completed within 3 days, and untargeted metagenomic sequencing was performed on the Illumina platform immediately, and yielded 2 × 150-bp paired-end reads. To avoid unexpected sequences, uninfected human CSF samples serving as controls were prepared in parallel, underwent library preparation, and were sequenced in the same run . All samP < 0.05). edgeR recommends filtering according to the count per million (CPM) value, and species with a CPM value greater than 1 are retained. Then, the number of read counts was converted into log2 CPM (log CPM), and the appropriate observation-level weights were calculated by estimating the mean-variance relationship, which was followed along with linear modeling. We used MEGAN6, a comprehensive toolbox for interactively analyzing microbiome data  Patterns of blood microbial composition in patients with bacterial meningitis. (B) Patterns of microbial composition of cerebrospinal fluid in patients with bacterial meningitis. Download Copyright © 2021 Liao et al.2021Liao et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


Can oxygenated portable normothermic perfusion of deceased donor livers for transplant improve outcomes compared with the current standard of care using ischemic cold storage?In this multicenter randomized clinical trial of 300 recipients of liver transplants with the donor liver preserved by either normothermic perfusion or conventional ischemic cold storage, normothermic machine perfusion resulted in decreased early liver graft injury and ischemic biliary complications and greater organ utilization.In this study, portable normothermic oxygenated machine perfusion of donor liver grafts resulted in improved outcomes after liver transplant and in more livers being transplanted. Ischemic cold storage (ICS) of livers for transplant is associated with serious posttransplant complications and underuse of liver allografts.To determine whether portable normothermic machine perfusion preservation of livers obtained from deceased donors using the Organ Care System (OCS) Liver ameliorates early allograft dysfunction (EAD) and ischemic biliary complications (IBCs).This multicenter randomized clinical trial  was conducted between November 2016 and October 2019 at 20 US liver transplant programs. The trial compared outcomes for 300 recipients of livers preserved using either OCS (n = 153) or ICS (n = 147). Participants were actively listed for liver transplant on the United Network of Organ Sharing national waiting list.Transplants were performed for recipients randomly assigned to receive donor livers preserved by either conventional ICS or the OCS Liver initiated at the donor hospital.The primary effectiveness end point was incidence of EAD. Secondary end points included OCS Liver ex vivo assessment capability of donor allografts, extent of reperfusion syndrome, incidence of IBC at 6 and 12 months, and overall recipient survival after transplant. The primary safety end point was the number of liver graft–related severe adverse events within 30 days after transplant.P = .01). The OCS Liver preserved livers had significant reduction in histopathologic evidence of ischemia-reperfusion injury after reperfusion . The OCS Liver resulted in significantly higher use of livers from donors after cardiac death . The OCS Liver was also associated with significant reduction in incidence of IBC 6 months  and 12 months  after transplant.Of 293 patients in the per-protocol population, the primary analysis population for effectiveness, 151 were in the OCS Liver group , and 142 were in the ICS group . The primary effectiveness end point was met by a significant decrease in EAD (27 of 150 [18%] vs 44 of 141 [31%]; This multicenter randomized clinical trial provides the first indication, to our knowledge, that normothermic machine perfusion preservation of deceased donor livers reduces both posttransplant EAD and IBC. Use of the OCS Liver also resulted in increased use of livers from donors after cardiac death. Together these findings indicate that OCS Liver preservation is associated with superior posttransplant outcomes and increased donor liver use.NCT02522871ClinicalTrials.gov Identifier:  This multicenter randomized clinical trial compares the use of oxygenated portable normothermic machine perfusion of deceased donor livers for transplant vs conventional ischemic cold storage to assess liver function preservation and outcomes among recipients of a liver allograft. The organs from donors with advanced age, multiple comorbidities, or donation after circulatory death (DCD) status are frequently declined for transplant owing to the clinician’s concern for increased risk of early allograft dysfunction (EAD), primary nonfunction, or serious chronic complications, such as ischemic biliary complications (IBCs).10Liver transplant provides lifesaving treatment for patients with end-stage liver disease. An inadequate supply of suitable donor organs has resulted in prolonged waiting times and high waiting list mortality rates.16Historically, advances in organ preservation have been linked to improved posttransplant outcomes and improved donor organ use. The concept of normothermic blood-based liver perfusion was developed to overcome the limitations of ICS.The multicenter International Randomized Trial to Evaluate the Effectiveness of the Portable Organ Care System (OCS) Liver for Preserving and Assessing Donor Livers for Transplantation  is the first US randomized clinical trial for liver perfusion and was designed to overcome the limitations of ICS, limiting the period of ischemia and providing physiologic assessment of liver graft function. The PROTECT trial prespecified donor characteristics from donation after brainstem death (DBD) and DCD donors that are known to be more vulnerable to ICS associated damage.CONSORT) reporting guideline. All participants gave written informed consent that was obtained in a manner consistent with the Common Rule requirements. No one received compensation or was offered any incentive for participating in this study.The PROTECT trial, a pivotal multicenter randomized clinical trial, compared posttransplant outcomes for recipients who received donor livers preserved using ICS or the OCS Liver  40 years of age or older; (2) expected total cross-clamp/cold ischemic time of 6 or more hours; (3) DCD donors if 55 years or younger; or (4) macrosteatotic livers (≤40%). Donor liver exclusion criteria included living donors, split livers, livers requiring accessory vessel reconstruction, or moderate to severe traumatic liver injury. Recipient exclusion criteria included younger than 18 years, acute or fulminant liver failure, prior solid organ or bone marrow transplant, chronic kidney failure, multiorgan transplant, ventilator dependence, or hemodynamic compromise.The OCS Liver consisted of an integrated system of 3 components: the OCS Liver console, OCS Liver perfusion set, and OCS bile salt solution for infusion A. The OCAfter organ acceptance, potential liver recipients who had provided informed consent for participating in the PROTECT trial were randomized to OCS Liver or to ICS preservation of the donor liver before the recovery team departed for organ retrieval. During organ recovery, once donor livers were examined and accepted for transplant and for inclusion in the PROTECT trial, preservation followed the randomization assignment. For the OCS Liver instrumentation, the hepatic artery and portal vein were cannulated for inflow, and the suprahepatic cava for outflow; the common bile duct was cannulated for bile collection and quantitation. Type-specific or O Rh-negative blood was sourced from banked irradiated, leukocyte-depleted packed red blood cells.Formalin-fixed biopsy material was received by the central laboratory, paraffin embedded, and sectioned at 4 µm prior to routine staining with both hematoxylin-eosin and CD31  immunostaining protocols. Sections were assessed for sectioning and staining quality before being converted to high-resolution  whole-slide imagery using a Zeiss AxioScan.Z1 automated scanning system. Resultant whole-scan images were quality controlled for focus and scanning artifacts and entered into a 21 CFR Part 11 compliant imaging database for prospective scoring (treatment group and donor organ subtype were not available to the reviewing pathologist). In total, 42 histopathologic criteria were electronically recorded in the database, which included comprehensive semiquantitative assessment of severity and composition of portal, lobular, and any other notable features. We used CD31 staining to assess the integrity of the endothelium lining. At the completion of the study, all histopathologic scoring was extracted and analyzed using R, version 3.6.3  for graphical and statistical data review, summary, and presentation.10 defined as the presence of 1 or more of the following: aspartate aminotransferase level higher than 2000 IU/L  within the first 7 postoperative days; bilirubin 10 mg/dL or higher  on postoperative day 7; international normalized ratio of 1.6 or higher on postoperative day 7; or graft primary nonfunction within the first 7 days, defined as irreversible graft dysfunction leading to recipient death or emergency retransplant, in the absence of immunologic or surgical causes.The primary effectiveness end point was the incidence of EAD,The secondary effectiveness and other critical clinical end points included the ability of the OCS Liver to monitor donor liver function throughout preservation; patient survival at day 30 or at initial hospital discharge if longer than 30 days; incidence of IBC defined as nonanastomotic ischemic strictures or bile leaks, confirmed with an endoscopic retrograde cholangiopancreatography or magnetic resonance cholangiopancreatography radiologic examination and blindly adjudicated by a 3-member independent clinical events committee; extent of reperfusion syndrome after transplant assessed by recipient’s lactate levels approximately 120 minutes after reperfusion in the recipient; and histologic assessment of the donor liver after transplant compared with baseline histologic specimen obtained before retrieval from the donor. All histopathologic evaluations were assessed using a blinded, independent core laboratory with extensive experience in liver transplant pathology.The primary safety end point was the mean number of liver graft–related severe adverse events (LGRSAEs) per patient within the initial 30 days after liver transplant. The LGRSAEs were predefined as a primary nonfunctioning graft, IBCs, hepatic vascular complications, or liver graft infection.The primary analysis population for effectiveness was the per-protocol population, consisting of all randomized recipients who received a donor liver that underwent the complete preservation procedure as per randomization assignment and had no major protocol violations. Safety was analyzed based on the as-treated population, consisting of all patients with a liver allograft. The modified intention-to-treat population consisted of all randomized patients who underwent a transplant in the PROTECT trial.The PROTECT trial was designed to test for both noninferiority and superiority if noninferiority was met. The primary effectiveness end point was analyzed by calculating the sample proportion of patients meeting the primary effectiveness end point, as well as an exact (Clopper-Pearson) 95% CI for the corresponding population proportion. The 95% upper bound of the exact unconditional 1-sided CI based on the Farrington-Manning score was calculated for the difference between the 2 population proportions (OCS − ICS). An upper confidence limit lower than δ = 0.075 would result in rejection of the null hypothesis in favor of the alternative hypothesis and the demonstration of noninferiority of OCS to ICS for the primary effectiveness end point. In the event noninferiority was demonstrated, the Fisher exact test (2-sided) was used to assess for superiority. The secondary end points were analyzed in a manner analogous to the primary effectiveness end point.t test with an α level of .05. Statistical analyses were conducted using SAS Studio, version 9.4 (SAS Institute Inc).The safety end point was analyzed by treatment group using descriptive statistics. For the mean number of LGRSAEs, the hypothesis was that the OCS Liver treatment was noninferior to the standard of care treatment, with a noninferiority margin of 1.00. The safety end point was analyzed using a 1-sided, 2-sample Between November 2016 and October 2019, a total of 429 patients provided informed consent to participate in the PROTECT trial, and 300 patients were randomized and received a liver allograft (153 in the OCS Liver group and 147 in the ICS group) . One recP < .001), despite the OCS Liver having significantly longer total cross-clamp (out of body) time  (P < .001). Overall, 155 DBD and DCD donor livers were assessed using the OCS Liver. Of those, 152 donor livers (98%) were successfully transplanted and analyzed in the PROTECT Trial. The 3 donor livers that failed OCS Liver assessment were from DCDs; 2 livers were rejected owing to lactate levels that continued to increase and reached higher than 10 mmol/L despite target flow parameters (P = .007)  of 276.6 (117.4) minutes and were maintained in near physiologic condition as shown by decreasing lactate levels from the start  to the end of perfusion   . A similar effect was seen in the modified intention-to-treat population (P = .004), a marker of ischemia-reperfusion injury19 (P = .39). We examined the clinical effect of reducing EAD by stratifying all trial recipients outcomes by presence or absence of EAD. We found that EAD was associated with significantly higher risk of graft failure compared with no EAD (log-rank test P = .003) (P = .04) and significantly longer overall hospital stay   A, signifP = .02) A and B.P < .001), and at initial hospital discharge, patient survival was 98.7% (149 of 151) for the OCS Liver group vs 98.6% (140 of 142) for the ICS group (noninferiority P < .001). The OCS Liver was associated with a significant decrease in ischemic biliary complications 6 months  and 12 months  after transplant (P = .046).20 There was no difference in the incidence of anastomotic nonischemic biliary complications . The type of LGRSAEs observed within the initial 30 days after transplant revealed an apparent decrease for the OCS Liver vs ICS in both IBCs (0 vs 2 events in 2 patients) and vascular complications (8 events in 7 patients vs 11 events in 9 patients)  number of LGRSAEs within the initial 30 days after transplant for the OCS Liver was noninferior to ICS  protocol recently showed impressive reduction in EAD and prevention of IBC in DCD liver transplant.The PROTECT trial was designed to overcome the significant limitations of ICS and to evaluate the clinical effect of reducing ischemic injury and providing functional assessment of liver allografts using a portable normothermic perfusion system. The use of the OCS Liver resulted in a significant reduction in ischemic injury associated with ICS and led to superior short-term and midterm clinical outcomes, with significant reduction of EAD and IBC through 12 months’ follow-up. The PROTECT trial provided clear mechanistic corroboration of the marked reduction of EAD observed in the trial based on the significant decrease in the histologic marker of ischemia-reperfusion injury, lobular inflammation, observed on histologic specimens recovered following reperfusion of the graft in the recipient, and significant mitigation of reperfusion syndrome in the recipient after transplant in the OCS group. The clinical importance of reducing EAD was reflected in significantly shorter ICU and hospital stays, and significant improvement in posttransplant graft survival. The PROTECT trial met its safety end point and reported low rates of LGRSAEs.33The PROTECT trial targeted DCD donors and DBD donors having risk factors that included older age, moderate level of steatosis, or anticipated long cross-clamp time, that portend EAD, primary nonfunction, or IBC. Use of the OCS Liver resulted in significantly increased DCD donor use as compared with ICS. These data suggest that the OCS Liver provided additional opportunity for ex vivo clinical optimization and assessment of the DCD liver graft, resulting in a higher proportion of DCD livers being successfully transplanted with the use of the OCS Liver compared with ICS. These results confirm the inherent clinical benefits of normothermic machine perfusion to provide an additional clinical quality assessment of liver allografts. Increasing use of DCD and questionable DBD liver allografts by demonstrating their viability and suitability for transplantation will likely be the greatest clinical benefit of ex vivo perfusion in liver transplant. The portability of the OCS Liver will likely gain increasing importance as organ retrieval teams travel further to recover organs with recently adopted allocation rules that mandate broader sharing of donor livers in the US.Complete blinding of the clinical teams to the preservation modality was impossible given the complex logistics involved in the application of machine perfusion at a remote site. Therefore, the randomization step was designed to occur only after the clinical decision of accepting the donor offer had been made based on clinical facts about the donor and without any knowledge of the preservation modality. If for any reason, the donor liver was rejected for transplant on physical examination during organ retrieval, the protocol prespecified that the potential recipient be rerandomized after a second donor liver was offered and clinically accepted. This process was established to ensure that clinical decision-making was the primary driver for accepting donor liver allografts and was not influenced by preservation modality. The balanced donor and recipient characteristics and risk factors as well as the equivalent baseline donor liver pathology assessment validate the robustness of this randomization process.The PROTECT trial showed superior short-term and mid-term clinical outcomes and higher numbers of donor livers used for transplant. The advent of portable, extracorporeal, donor liver machine perfusion offers for the first time a convenient and effective approach to both assess and enhance donor liver function, thereby improving transplant safety, expanding the liver donor pool, and reducing waiting list mortality.