Mesenchymal stem/stromal cells (MSCs) are widely distributed in the body and play essential roles in tissue regeneration and homeostasis. MSCs can be isolated from discarded tissues, expanded in vitro and used as therapeutics for autoimmune diseases and other chronic disorders. MSCs promote tissue regeneration and homeostasis by primarily acting on immune cells. At least six different types of MSCs have been isolated from postnatal dental tissues and have remarkable immunomodulatory properties. Dental stem cells (DSCs) have been demonstrated to have therapeutic effects on several systemic inflammatory diseases. Conversely, MSCs derived from nondental tissues such as the umbilical cord exhibit great benefits in the management of periodontitis in preclinical studies. Here, we discuss the main therapeutic uses of MSCs/DSCs, their mechanisms, extrinsic inflammatory cues and the intrinsic metabolic circuitries that govern the immunomodulatory functions of MSCs/DSCs. Increased understanding of the mechanisms underpinning the immunomodulatory functions of MSCs/DSCs is expected to aid in the development of more potent and precise MSC/DSC-based therapeutics. In almost every vertebrate organ, there are mesenchymal stromal cells (MSCs). Because these cells possess the capacities of self-renewal and are progenitors that can give rise to adipocytes, chondrocytes, osteoblasts and myofibroblasts in response to differentiation or inflammatory cues, they are frequently referred to as mesenchymal stem cells . Due to their multilineage differentiation potential, ability to regulate the immune response, easy accessibility to source tissues and excellent propagation capacity in vitro, MSCs have been regarded as an ideal source for therapeutics in tissue regeneration and autoimmune and hyperinflammatory diseases. MSCs are a highly heterogeneous cell type with multiple postnatal tissue origins. These cells originate either from the mesoderm or ectoderm –4. The dTo date, at least six different types of postnatal dental tissue-derived MSCs have been isolated and characterized, including stem cells from dental pulp (DPSCs), periodontal ligament (PDLSCs), deciduous teeth (SHED), apical papilla (SCAPs), dental follicles (DFSCs), and gingiva (GMSCs) Fig. . STRO-1 + pericyte-derived DSC precursors contributed to odontoblast differentiation during dental pulp development and tissue regeneration [+ MSCs surrounding the cervical loop of the incisor are the progenitor cells of PDLSCs and DPSCs, and Gli1+ cells within the suture mesenchyme are the major MSCs responsible for craniofacial bone development and homeostasis [+ SCAPs are multipotent tooth root stem cells that can regenerate tooth roots with functional dentin and neurovascular-like structures [MSCs residing in most internal organs are primarily derived from the mesoderm. DSCs originate from the neural crest. Despite their distinct origins, MSCs/DSCs all possess general properties such as self-renewal, multipotent differentiation, immunomodulation, and the expression of a set of MSC surface markers . Howeverneration , 13. Genneration . Zhao eteostasis , 16. CD2ructures . These sDSCs exhibit increased odontogenic preference but have reduced chondrogenic and adipogenic differentiation potential . The difDSCs can be isolated from different parts of the tooth, and accumulating evidence has shown functional differences among DSCs in terms of proliferation rate, osteo/odonto differentiation rate and angiogenic potential . TherefoPrevious studies have shown that the regenerative and immunomodulatory activity of DSCs is dynamically regulated by the stem cell niche and immune microenvironment , 24. PDLDSCs exert immunosuppressive effects mostly through the secretion of immunoregulatory cytokines, which modulate innate and adaptive immunity, as well as the complementary system . Thus, tCompared with mesoderm-derived MSCs, neural crest-derived MSCs show increased differentiation potential to neural cells and chondrocytes, as well as increased immunomodulatory and anti-inflammatory effects in vitro and in vivo, suggesting that DSCs have great therapeutic potential for applications in tissue repair, including bone, dental and soft tissues .via an endochondral bone ossification-like process [A previous study demonstrated that PDLSCs showed a stronger tissue regeneration capacity than MSCs derived from bone marrow (BM-MSCs) on critical-size defects in the immunodeficient rat calvarium, suggesting the superiority of using PDLSCs in bone tissue regenerative therapy . The tra process . These sIn dental tissue repair, DSCs can generate dental-related tissue after transplantation in vivo. Gronthos et al. reported that DPSCs could regenerate a dentin-pulp complex, which is composed of mineralized matrix and fibrous tissue . Functio+ DPSCs could survive for 1 month after transplantation in the brains of nude mice and form complete blood vessels that integrated into the host cerebrovascular system [In addition to their applications in bone regeneration, DSCs are also recommended for soft tissue reconstruction, such as in periodontal ligament, dental pulp and nerve tissue –43. Becar system .The immunomodulatory property of DSCs makes them suitable therapeutics for aberrant immune and inflammatory diseases. DSCs can exert their immunomodulatory effects through direct and indirect mechanisms , 51. DPSThe administration of SHED-derived CM reduces myocardial infarct size and inflammatory cytokine levels, such as TNF-α, IL-6, and IL-β . The antSince the safety and efficacy of DSCs have been extensively verified, DSCs are increasingly being used in clinical studies. Currently, there are more than 15 clinical trials for tissue regeneration and disease repair using DSCs  in rodents  or indoelmine-2–3-dixoygenase (IDO) in other mammalian species to suppress T-cell responsiveness , 69. Murferation . Moreoveferation . Howeverferation .+ T cells toward differentiation into Treg cells [The immunosuppressive effects of human MSCs in response to stimulation with IFN-γ plus TNF-α or IL-1 primarily rely on IDO. IDO is a catabolic enzyme that degrades tryptophan to kynurenine. Depletion of local tryptophan by IDO activates the stress-response kinase general control nonderepressible 2 (GCN2), which senses amino acid withdrawal. GCN2 activation inhibits T-cell proliferation and biases naive CD4eg cells , 74. Moreg cells  and indueg cells .+/CD206+ macrophages in a rat model of periodontitis [NFE2L2 in macrophages. NFE2L2-encoded NRF2 not only functions as a master regulator of antioxidant defense but also represses the expression of inflammatory genes. As expected, NRF2 upregulation in macrophages was inhibited by inhibiting IDO and 1-methyltryptophan (1-MT), and the anti-inflammatory effect of ADSCs on macrophages was blocked when NRF2 expression in macrophages was silenced. Kynurenic acid, another IDO-derived metabolite that shares the same aryl hydrocarbon receptor as kynurenine, can promote TNF-α-stimulated gene-6 (TSG-6) expression and alleviate neutrophil infiltration in injured lungs [A recent study showed that local injection of ADSCs significantly reduced alveolar bone loss and the ratio of iNOSdontitis . IDO exped lungs .In addition to iNOS and IDO, the immunosuppressive properties of MSCs are also mediated through the expression of COX1/COX2 enzymes and the production of prostaglandin E2 (PGE2). While COX1 is constitutively expressed in most mammalian cells, COX2 is an inducible enzyme and cab ne induced by proinflammatory cytokines and growth factors . PGE2 haDSCs exert their regulatory effects on inflammation similar to MSCs derived from other tissues. GMSCs specifically suppress peripheral blood lymphocyte proliferation and induce the expression of a wide range of immunosuppressive factors, including IDO, iNOS, and COX-2, in response to inflammatory cytokines . PGE2 plMSCs secrete a variety of growth factors, which not only function as effector molecules during tissue repair but also regulate the differentiation of MSCs themselves. During adipose tissue injury and repair, fibroblast growth factor-2 strongly promotes ADSC proliferation and HGF secretion through the c-Jun N-terminal kinase signaling pathway. HGF expression in MSCs contributes to the regeneration of adipose tissue and suppression of fibrogenesis after injury . The secMSCs can produce insulin-like growth factor 2 (IGF-2) to preprogram macrophages to undergo anti-inflammatory polarization during their maturation . When exSimilar to BM-MSCs, which are capable of reprogramming macrophages into the M2 phenotype , GMSCs c+ T-cell activation by suppressing STAT3 phosphorylation and reversing symptomatic neuroinflammation in experimental autoimmune encephalomyelitis [+ macrophages in vitro, and CCL2 expression by MSCs is required for IL-10-mediated polarization of intestinal and peritoneal resident macrophages in vivo [The chemokines expressed by MSCs have been reported to regulate the migration and function of immune cells to maintain tissue homeostasis. BM-MSCs can rapidly express MCP1 in response to circulating TLR ligands or bacterial infection and induce monocyte trafficking into the bloodstream . BM-MSC-myelitis . CCL2 an in vivo . We also in vivo .MSCs also exert their therapeutic effects through the secretion of EVs. EVs are lipid-bound vesicles that are secreted into the extracellular space. The cargo of EVs consists of proteins, organelles and nucleic acids, which are responsible for the regulatory function of MSCs. The main subtypes of EVs include exosomes (50–100 nm in diameter), microvesicles , and apoptotic bodies (1 μm to 5 μm in diameter). MSCs have been shown to secrete at least 3 types of EVs, which can be differentially isolated based on their affinities for membrane lipid binding ligands and are likely to have different biogenesis pathways and different functions .MSC-derived EVs have been shown to exhibit potent immunoregulatory properties. For example, human adipose MSC-derived exosomes inhibit the differentiation and activation of T cells and reduce T-cell proliferation and IFN-γ release . The exoDSCs can also produce EVs to promote immune homeostasis. GMSCs even have a higher yield of small EVs than BM-MSCs and skin MSCs . These svia arrestin domain-containing protein 1-mediated microvesicles. The vesicles are then engulfed and reused by macrophages, resulting in enhanced macrophage bioenergetics and impaired TLR signaling [MSCs undergo mitophagy in response to oxidative stress and promote the translocation of depolarized mitochondria to the plasma membrane ignaling . MSC EV-ignaling .Exosomal miR-182 delivery from BM-MSCs to macrophages converted inflammatory macrophages to the M2 phenotype by targeting TLR4, reducing inflammation levels in a mouse model of myocardial ischemia/reperfusion .via electrostatic charge interactions. FasL in apoVs could mediate the cell death pattern switch in neutrophils from NETosis to apoptosis and then inhibit the migration of neutrophils from bone marrow to distal organs, alleviating organ injury and sepsis [During the treatment of immune diseases with MSCs, researchers found that despite being immunosuppressive, MSCs were undetectable after administration. Galleu. et al. found that MSCs were actively induced to undergo perforin-dependent apoptosis in a murine graft-versus-host disease model and that their apoptosis is essential to initiate MSC-induced immunosuppression. After infusion, recipient phagocytes engulf apoptotic MSCs and produce IDO, which mediates the immunosuppressive function of MSCs . In addid sepsis .via the FasL-dependent Fas pathway. Fas and FasL are members of the tumor necrosis factor (TNF)-receptor and TNF family, respectively. The binding of Fas to FasL results in the activation of a caspase cascade that initiates apoptosis. Apoptotic T cells subsequently trigger macrophages to produce high levels of TGF-β, which in turn leads to an increase in regulatory T cells and immune tolerance [It has been reported that BM-MSCs induce T-cell apoptosis olerance . DPSCs colerance . Moreoveolerance .A landmark paper by Ren et al. demonstrated that the immunomodulatory properties of murine MSCs were not innate but were elicited by a set of inflammatory cytokines  Fig. 3)3). T-celWhile the aforementioned inflammatory cytokines are critical in inducing the immune modulatory properties of MSCs, the anti-inflammatory cytokine TGF-β has the opposite effect on the immunosuppressive effect of MSCs on anti-CD3-activated splenocytes. TGF-β can inhibit iNOS expression in a SMAD3-dependent manner. TGF-β produced by MSCs can act in an autocrine manner to reduce iNOS .Single-cell transcriptome analysis of human oral mucosa in healthy individuals and patients with periodontitis was recently reported . The tralpr and Caspase 3−/− mice, the self-renewal and osteogenic/adipogenic differentiation of MSCs was significantly impaired. The infusion of exogenous apoptotic bodies rescued these impairments and ameliorated the osteopenia phenotype in MRL/lpr, Caspase 3−/− and ovariectomized mice. MSCs could engulf apoptotic bodies via integrin αvβ3 and reuse the apoptotic body-derived ubiquitin ligase RNF146 and miR-328-3p to inhibit Axin1 and thereby activate the Wnt/β-catenin pathway [In the human body, billions of cells undergo apoptosis every day, and a large number of apoptotic bodies need to be cleared to maintain tissue homeostasis . Apoptot pathway . In addi pathway . The apoAs discussed previously, MSCs produce abundant levels of EVs to influence immune cells and other types of cells. It is likely that MSCs may also take up circulating vesicles and change their function and behaviors accordingly. The metabolites released by vesicles, as well as those produced from other routes, could have an impact on the immunomodulatory functions of MSCs. Future studies should provide more insights into the effects of vesicles on MSCs.Mechanical force plays a critical role in determining the fate and characteristics of PDLSCs. Alveolar bone osteocytes produce sclerostin, a Win inhibitor, to negatively regulate Gli1+ PDLSC activity. Sclerostin expression is inhibited by physiological occlusal force . Force-tA recent study showed that MSCs on a soft matrix produce higher levels of immunomodulatory factors than those on a stiff matrix . The expAnisotropic silk protein nanofiber-based hydrogels were developed to mimic the physical microenvironment inside the blastocele and were more effective in sustaining the stemness of mouse embryonic stem cells (mESCs) than the classical recipe containing leukemia inhibitory factor and mouse embryonic fibroblasts (MEFs) . The mESThe metabolic pathways required for MSCs to acquire and sustain their immunoregulatory functions have been studied recently. Inflammatory cytokines cause metabolic shifts in MSCs, including enhancing glycolysis and increasing fatty acid oxidation, but only interference with glycolysis but not fatty acid oxidation impairs IDO upregulation and the immunosuppressive function of MSCs , 136. Th2+ transfer from the ER to mitochondria, and mitochondrial calcium overload further damages mitochondria. Due to the inhibition of mitophagy under chronic inflammatory conditions, damaged mitochondria continuously accumulate in MSCs [2+ around mitochondria reduced mitochondrial calcium flux, physiologically restoring the function of mitochondria and MSCs.Maintaining proper mitochondrial turnover and function is critical for cellular homeostasis and many biological processes. It is well recognized that disrupted mitochondrial homeostasis contributes to neural degeneration. Likewise, mitochondrial dysfunction may compromise the functions of MSCs and lead to chronic inflammation-associated bone diseases, such as periodontitis and osteoarthritis. It was recently shown that chronic inflammation leads to excess Ca in MSCs . NanoparWhile transplanted MSCs have been widely documented to exert potent immunomodulatory effects in various autoimmune diseases and graft-versus-host disease (GvHD), MSCs are rapidly cleared in recipients after their administration. The dynamics of transplanted MSCs in recipients indicate that MSCs do not need to persist very long to exert their therapeutic effects. It appears that their only mission is to deliver signals or messages to the recipients or to awaken host tissues. Using a murine model of GvHD, Dazzi and colleagues observed that MSCs actively underwent perforin-dependent apoptosis, which is essential for MSCs to have immunosuppressive functions . In GvHDWith increased understanding of the mechanisms by which MSCs/DSCs exert their immunoregulatory effects and the extrinsic and intrinsic factors that stimulate and sustain the immunoregulatory functions of MSCs, it is possible to manipulate MSCs by various means to achieve enhanced therapeutic efficacies. We discuss some of the strategies that can increase MSC potency.Like immune cells that need to be activated by intrinsic or extrinsic signals to achieve their mission, MSCs also need to be activated or induced to acquire their immunomodulatory function. Inflammatory cytokines produced by immune cells or other types of cells serve as essential stimuli to induce immunomodulatory functions . Thus, MGlycolysis is critical for the inflammatory function of various immune cells, and blocking glycolysis using 2-deoxy-D-glucose (2-DG) greatly compromises the proinflammatory response of immune cells. However, unlike in immune cells, this metabolic pathway serves to support the immunosuppressive function of MSCs. To behave as immunomodulators, MSCs need to be switched to a massive biosynthesis mode to produce a large number of chemokines and effector molecules in large quantities. Glucose understandably provides the carbon resources that are in great demand. Therefore, any means that can boost glycolysis in MSCs may increase the production of factors by MSCs. Because MSCs and inflammatory immune cells may compete for glucose in the tissue microenvironment, strategies that block glycolysis in immune cells but favor glucose uptake and glycolysis in MSCs will likely enhance their immunomodulatory functions. Metabolites derived from glycolysis, such as lactate, may also participate in the immunomodulatory function of MSCs. Mitochondria, where some crucial metabolic activities take place, may also contribute to the regulation of MSC immunomodulatory functions, and the exact roles may be elucidated in future studies.Since the functions and fates of MSCs are determined by various biophysical cues they sense, it is possible to amplify the therapeutic functions of MSCs by implanting special MSC-laden scaffolds. MSCs seeded on certain matrices may possess functions that are not present in MSCs in monolayer cultures and last longer in vivo. An emerging field called developmental tissue engineering mimics morphogenetic processes during development to harvest biofunctional tissues and organs . These a


Dimerization of TF effectively sequesters mostof the substrate binding sites, which are expected to become accessible uponbinding to the ribosome as a monomer. As TF lacks ATPase activity, which iscommonly used to trigger conformational changes within molecular chaperonesin action, the ribosome-binding-associated disassembly and conformationalrearrangements may be the underlying regulatory mechanism of its chaperoneactivity.Trigger factor (TF) is a highly conserved multi-domain molecularchaperone that exerts its chaperone activity at the ribosomal tunnel exitfrom which newly synthesized nascent chains emerge. TF also displayspromiscuous substrate binding for a large number of cytosolic proteinsindependent of ribosome binding. We asked how TF recognizes a variety ofsubstrates while existing in a monomer–dimer equilibrium. Paramagneticnuclear magnetic resonance (NMR) and electron spin resonance (ESR)spectroscopy were used to show that dimeric TF displays a high degree ofstructural polymorphism in solution. A series of peptides has been generatedto quantify their TF binding affinities in relation with their sequencecompositions. The results confirmed a previous predication that TFpreferentially binds to peptide fragments that are rich in aromatic andpositively charged amino acids. NMR paramagnetic relaxation enhancementanalysis showed that TF utilizes multiple binding sites, located in thechaperone domain and part of the prolyl  TF is a unique molecular chaperone in that it is the first molecular chaperone that all newly synthesized nascent polypeptide chains encounter . TF binds to the ribosomal protein L23 through the RBD in aMolecular chaperones are pivotal in facilitating protein folding and maintaining proteostasis in vivo . In prokaryotes, trigger factor (TF) is a highly conserved multi-domain molecular chaperone, consisting of a ribosome binding domain (RBD),substrate binding domain (SBD), and a prolyl peptidyl E. coli proteome that require the contributions of TF to fold correctly . Although the number of obligatorily substrates of TF within the E. coli proteome is limited, TF playsan important role in working with SecA and SecB to regulate the membraneprotein secretory pathway, as nascent membrane proteins need to be correctlysorted by TF and SecA/B as they emerge from the ribosome . It therefore raises the question as to how TF recognizes specific substrates when faced with a multitude of sequence variations of the bacterial proteome during co-translational and post-translational folding. To this end, an empirical scoring function for predicting TF binding motifs has been proposed based on peptide array analyses: a putative TF binding motif should be at least eight amino acids in length and contain both aromatic  and positively charged lysine or arginine residues . In a landmark study, Kalodimos and co-workers demonstrated that it requires threeTF molecules to bind to one fully unfolded PhoA, which contains multiple TFbinding sites with low-micrometer binding affinities . TFexhibits multiple substrate binding sites in SBD and PPI, which areevolutionarily conserved. The additivity of substrate binding affinities in the multiple binding sites on SBD and PPI results in much higher binding affinity and specificity for long PhoA fragments with an apparent Kd in the nanometer range. The strong binding enables the determination of the solution structures of TF in complex with the three different PhoA fragments by solution-state NMR spectroscopy complex. The structural information of TF incomplex with various PhoA fragments indicated that TF preferentially bindsto aromatic residues as well as large hydrophobic residues. There is noindication of the preference for positively charged lysine or arginineresidues as previously predicted.Although TF primarily acts on the ribosome in a co-translational manner, TFhas been shown to exhibit chaperone activity in the absence of the ribosometo promiscuously facilitate folding of cytosolic proteins. The crystalstructure of a thermophilic TF in complex with the ribosomal protein L9shows that TF binds to its substrate in a In this study, we sought to evaluate the predictive power of the empiricalscoring function for TF binding motifs proposed by Bukau and co-workers. Combining methyl NMR and electron spin resonance (ESR) spectroscopy, we confirmed the dynamic and polymorphic nature of the TF dimer. We generated a collection of fluoresceinisothiocyanate (FITC)-labeled peptide to validate by fluorescencepolarization (FP) the predictive power of the proposed TF binding scoringfunction. Two TF binding peptides were subsequently spin-labeled forparamagnetic relaxation enhancement (PRE) measurements to identify multiplesubstrate binding sites within the SBD and PPI. Importantly, we demonstratedthat dimerization of TF can sequester these binding motifs and that thedynamic equilibrium between monomer and dimer is essential for substraterecognition. Collectively, our findings illustrated the functionalimportance of TF dimerization in the context of co-translational folding.22.1E. coli TF was obtained from the Nara E. coli ORF collection  and had beensubcloned into a pET-21d plasmid with a HisE. coli DH5The open reading frame of E. coli culture in perdeuterated M9 medium containing 99.9 % Unlabeled, uniformly The cells were harvested by centrifugation using a Beckmann J20XP centrifugewith a JLA 8.1K rotor for 30 min with 6000 rpm at 4 2.2Five peptides corresponding to fragments of isocitratedehydrogenase  were synthesizedin-house. A fraction of all these peptides were subsequently labeled withFITC at the N-terminus for fluorescence polarization (FP) measurements. Allpeptides (with and without FITC labeling) were purified by high-performanceliquid chromatography (HPLC) and validated by MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) mass spectrometryagainst their expected molecular weights. For FP analysis, FITC-labeledpeptides were dissolved in DMSO to yield a stock solution of 4 M. They weresubsequently diluted by 20 mM Tris (pH 7.4) and 100 mM NaCl to yield a molar concentration of 100 2.3https://nmrfam.wisc.edu/nmrfam-sparky-distribution/, last access: 2 June 2021). The nitroxide of MTSL was reduced by adding an aliquot of ascorbic acid to yield a final concentration of 1 mM. The observed PREs were expressed as the ratio of the peak intensities of the oxidized (paramagnetic state) to the reduced (diamagnetic state) state . Immediately after the removal of DTT, 10-foldmolar excess of MTSL was added, and themixtures were incubated overnight at 4 For double electron–electron resonance (DEER) measurements, samples were prepared either by single-labeled TFvariants or 3TF exists in solution in a monomer–dimer equilibrium with a low-micrometerTo further probe the dynamics of individual domains in the context of adimeric TF, we employed ESR spectroscopy with site-specific spin labels. Weindividually introduced a spin label to one of the four sites in TF, namelyresidue 14 on the RBD (14C), residue 150 on PPI (150C), and residues 326 or376 on the SBD (326C or 376C), by covalently attaching a MTSL to the mutatedcysteine side-chain . The spin-labeled TF variants wereanalyzed by CW-ESR to probe the domain dynamics manifested in the lineshapes. Comparison of the CW-ESR spectra the TF variants showed distinctside bands for 14C at 310 K, suggesting the presence of multipleconformations . In contrast, the CW-ESR spectrum of 326C showedminor signals that were similar to those of 14C, while 376C did not exhibitthe same signals, implying that the conformational heterogeneity of the RBDis more pronounced than that of the SBD. In the case of 150C, there was noindication of conformational heterogeneity as the ESR lines were sharpwithout a side band. ESE analysis was subsequently used to deduce thetransverse relaxation time , several surface-immobilized peptides showed prominentTF binding . Together with the results derived fromother peptide arrays, an empirical scoring function for predicting thepotential TF binding site along a given protein sequence was proposed . Nevertheless, the predictive power of such a scoring function has not been experimentally verified thus far. According to the prediction, an ideal TF binding motif should be at leasteight residues long and rich in aromatic residues and positively chargedlysine or arginine. The requirement for the coexistence of hydrophobic andcharged residues is an intriguing feature. Nevertheless, the relativelyloose definition can lead to a huge number of potential binding sites withinthe bacterial proteomes. As a model system, we correlated the previouslyreported peptide array data of TF binding to ICDH  and the predicated TF binding score as a function of ICDH sequence . By visual inspection of the blotting densities of the peptide array, weidentified five segments within the ICDH sequence that showed strong TFbinding and fulfilled the requirement of peptide length and composition(Table 1). We adjusted the window sizes of the selected sequences tomaximize the amount of preferred amino acid types and chemically synthesizedthese peptides followed by introducing a FITC moiety at the N-terminiindividual peptides to facilitate fluorescence polarization (FP)measurements to determine the binding affinities of these peptides to TF.Except for IcdH3 and IcdH4, whose sequences are partly helical in the crystalstructure , all the remaining sequences correspond to loop regions that do not adopt particular secondary structures. The resultingdissociation constants , which had an endogenous cysteine residuewithin their sequences that can be spin-labeled with MTSL for paramagneticrelaxation enhancement (PRE) measurements. We first usedU-[4In this study, we employed methyl NMR and ESR spectroscopy to characterizethe dynamics of full-length TF in its dimeric form. Although TF exists inequilibrium between monomer and dimer, the experimental conditions underwhich the NMR and ESR experiments were conducted, i.e., protein concentrations well above 0.25 mM, ensured that TF is predominantly dimeric, as has been established previously . Our findingsindicated that the TF dimer interface is not well defined, which may existin several distinct configurations, as evidenced by the severely broadenedlinewidths of the alanine methyl group within the RBD . Theconformational heterogeneity within the RBD also manifested in theadditional side bands of the CW-ESR spectra of 14C, which probe theenvironment around the MTSL moiety at the RBD . Furthermore, thereis a good correlation between the NMR methyl linewidth analysis and the T2analysis of the MTSL-labeled TF variants, which showed that the PPI is themost dynamic, consistent with the previous findings that TF forms anantiparallel dimeric assembly where PPI makes limited contacts with otherdomains .We next collected DEER data on singly MTSL-labeled TFs or We next generated five FITC-labeled peptides derived from ICDH todemonstrate the predicted power of the empirical scoring function for TFbinding based on the sequence composition . Two peptidesthat harbor an endogenous cysteine within the sequences, namely IcdH2 andIcdH3, were spin-labeled with MTSL to map their binding sites on TF by PREmeasurements. We identified three distinct binding sites within the SBD andone binding site within the PPI . The locations of thesebinding sites are consistent with the previous study in which four disordered fragments of PhoA are used to map the binding sites on TF . The authors also reported multiple binding sites within thePPI and SBD when short peptides were used to map the binding sites bychemical shift perturbations and intermolecular NOEs. When a longer peptidefragment of PhoA is used as a substrate, each of the substrate binding siteswithin the PPI and SBD is occupied by a specific TF binding motif, therebyleading to a unique binding mode that enables structure determination of thesubstrate-bound TF. By determining the microscopic According to the ESR analysis, the spin labels within the RBD and SBD aremostly solvent-exposed . Furthermore, the dimer interface appearedto be quite heterogeneous and dynamic, according to methyl NMR and CW-ESRline shape analyses  and the more robust DEER measurements .The unique domain architecture of TF suggests that the dimer interface doesnot form a properly encapsulated cavity to accommodate its substrates.Additionally, the distributions of sparsely negatively charged surfacessurrounded by small patches of neutral (hydrophobic) surfaces within the SBDand PPI coincide with the observed peptide binding sites, which may explain whypositively charged residues and aromatic residues are both favored for TFbinding. Unlike GroEL/GroES, which has an efficient nucleotide-dependentregulatory mechanism to mechanically control the exposure of its substratebinding sites, TF may utilize the self-dimerization to achieve the sameregulation .Here we compared the peptide-binding-induced PREs in PPI5The intricate interplay between TF, nascent chains and the ribosome can be modulated by the sequence compositions of the nascent chains. Our NMR, ESR, and biophysical analyses confirmed the structural polymorphism of the TF dimer and the multivalency of substrate binding, which is sensitive to TF dimer formation. These results led us to propose that the relatively strong ribosome binding affinity serves as the key regulatory mechanism to modulate monomer–dimer equilibrium and therefore the accessibility of the substrate binding sites, which are fully exposed when TF binds to the ribosome through its RBD. The observed binding affinities of the selected peptides from ICDH indeed fit well within the dynamic range of these binding events.10.5194/mr-2-375-2021-supplementThe supplement related to this article is available online at: https://doi.org/10.5194/mr-2-375-2021-supplement.


If the damaged plasmalemma is not rapidly repaired within minutes, activation of apoptotic pathways by calcium influx often results in cell death. We review publications reporting what is less-well known (and not yet covered in neuroscience or cell biology textbooks): that calcium influx at the lesion sites ranging from small nm-sized holes to complete axonal transection activates parallel biochemical pathways that induce vesicles/membrane-bound structures to migrate and interact to restore original barrier properties and eventual reestablishment of the plasmalemma. We assess the reliability of, and problems with, various measures  used individually and in combination to assess plasmalemmal sealing in various cell types . We identify controversies such as plug versus patch hypotheses that attempt to account for currently available data on the subcellular mechanisms of plasmalemmal repair/sealing. We describe current research gaps and potential future developments, such as much more extensive correlations of biochemical/biophysical measures with sub-cellular micromorphology. We compare and contrast naturally occurring sealing with recently-discovered artificially-induced plasmalemmal sealing by polyethylene glycol (PEG) that bypasses all natural pathways for membrane repair. We assess other recent developments such as adaptive membrane responses in neighboring cells following injury to an adjacent cell. Finally, we speculate how a better understanding of the mechanisms involved in natural and artificial plasmalemmal sealing is needed to develop better clinical treatments for muscular dystrophies, stroke and other ischemic conditions, and various cancers.Neuroscientists and Cell Biologists have known for many decades that eukaryotic cells, including neurons, are surrounded by a plasmalemma/axolemma consisting of a phospholipid bilayer that regulates trans-membrane diffusion of ions  and other substances. Cells often incur plasmalemmal damage  Eukaryotic cells in multicellular organisms often incur damage to their plasmalemma, the membranous diffusion barrier between the extracellular and intracellular fluids that completely surrounds each individual cell . The plaTraumatic damage to a plasmalemma ranges from a small hole of nanometer-to micron-sized diameters to complete transections of a cell body or its cytoplasmic extensions  that may be many  microns in diameter . Prior t2+) at the wound site that is required to initiate sealing. This influx of extracellular Ca2+ activates parallel biochemical pathways, intracellular organelles common to vesicular trafficking in the Golgi apparatus, conventional exocytosis, and coordinated exocytosis at chemical-releasing synapses . That is, sealing dynamics by vesicular interactions that occur after onset of plasmalemmal trauma take seconds to minutes in different cell types to restore a plasmalemmal seal having diffusion barrier properties observed for their originally-intact plasmalemma. These vesicular interactions could form a vesicular “plug” that produces a constantly changing diffusion barrier that gradually attains the barrier properties of the intact plasmalemma  and input resistance (Rin) are two measures used to assess the state of a membrane seal in a variety of cell types, although these measures have not been used in most recent studies  measures of the status of a membrane seal may be reasonably accurate for cells where all plasmalemmal extensions are less than a space constant from the intracellular recording and stimulating electrode(s). However, many cells (or long cytoplasmic extensions such as axons or dendrites) often have membrane cable properties distributed over many space constants. For these cells or cytoplasmic extensions, measurements of Vm and Rin are often very insensitive to changes in Rin at the lesion site, even when measurements are taken within 0.01 of a space constant from the transection or lesion site  do not depend upon axon cable properties. Hence, the extracellular measurement of Ii by a vibrating probe combined with intracellular measures of Vm provide a more reliable measure of the effectiveness of an axonal seal  axon. Measures of Iimust be paired with measures of Vm that show that Vm is equal to control values for the intact plasmalemma before the cell was injured  transmission electron microscopy (TEM) is needed to detect whether a membrane is continuous and intact. Even then, it is almost impossible to collect and analyze many serial sections and develop a 3D reconstruction of the membrane topology . To the jury See  using 50jury See  or 100X jury See  do not hm, Rin, and Iin. Additionally, vesicles may collapse or explode as a result of the dehydration and other processes applied during TEM or SEM sample preparation (Scanning electron microscopy (SEM) has also been used to observe the accumulation of vesicles at the transected ends of axons  and lesiparation .m and Iin, and high-resolution TEM images taken through the lesion site. These multiple-method measures of membrane repair have primarily been studied using invertebrate giant axons (GAs), namely, squid, crayfish, and earthworm GAs  to traumatic lesions than unfertilized eggs  of invertebrate nerve cells are often used as a model system to assess sealing of small holes or complete transections of membrane-bound structures. These cytoplasmic extensions do not have an abundance of vesicles docked at the membrane . Inverte of Ca2+ . Data fr of Ca2+ .Cultured mammalian PC12 or B104 cells as a model system do not exhibit instantaneous sealing and show progressive barrier formation , as may The use of damaged cells in culture to assess plasmalemmal sealing after traumatic injury involves shearing/scraping of single layer cell cultures  or syringe uptake of sea urchin eggs. In the former procedure, cells are grown to ∼70–90% confluency and the culture dish is scraped to induce widespread, unquantified plasma membrane damage to many cells . In the Prior to 1994, the subcellular, biochemical, etc., mechanisms that repair a damaged plasmalemma had not been directly studied. Lipid spreading was often assumed to repair small holes and collapse and fusion of apposed plasmalemmal leaflets were assumed to rapidly seal a complete transection resulting in a continuous plasmalemma whose chemical composition would be similar to the intact membrane .The sealing of micro-punctures in the plasma membrane were assumed to be repaired by lipid spreading . In thisLarger-scale plasmalemmal damage of transected cytoplasmic processes was originally assumed to seal by collapse and subsequent fusion of cut plasmalemmal leaflets  Kelly, , althougm and Ii recovered to values representative of a successful membrane seal. Accompanying low-resolution phase contrast microscopy was used to claim the establishment of a high resistance, partition-like bi-layered membrane proximal to the transected end  disassembly is correlated with Casmalemma . Followi G-actin . This re G-actin . MT-inde G-actin . For exa G-actin .2+ channels that comprise the cellular secretory machinery. For example, localization of the synaptic fusion protein SNAP-25  constricts disruptions by generating tensile forces in the surrounding plasmalemma  at a lesion site.2+ influx, along with (perhaps to a lesser extent) release of Ca2+ from internal sources , reactive oxygen species (ROS) and mechanical distortion/stress of the plasmalemma  to induce sealing in cultured rat hippocampal neurites , rather than post-injury  in C2C12 cell culture results in the development of filopodia-like structures  in the absence of Caructures , possiblructures  can induolutions .2+ influx following injury activates several phospholipases that influence sealing through parallel biochemical pathways -1 (Novel PKC (nPKC) and conventional PKC (cPKC) are stimulated by AA . Injured (Arf)-1 , a Rho-f (Arf)-1 . PA then (Arf)-1 . LPA rel (Arf)-1 . PIP2 cl2+ uniporter takes up excess Ca2+ following wounding. Increased Ca2+ produces a transient increase in the mitochondrial production of ROS  are produced by, or are affected by, cytoplasmic reactions and organelles . For exan of ROS . An incrn of ROS . Inhibityoblasts . RhoA reyoblasts . The intyoblasts , suggest2+. In cultured muscle cells, activation of Tri-Partite Motif (TRIM) family proteins is oxidation-dependent  and IP3 through cleavage of PIP2   plasmalemmal holes see . The cel2+ concentration upregulates cyclic AMP (cAMP) that activates both PKA and Epac1/Epac2  accumulates at sites of nanometer-sized plasmalemmal holes until sealing is complete  and is oand Vps4 . AnxA7 mand Vps4 . Filamenand Vps4 . ESCRT cand Vps4 , or by pand Vps4 .Pathways facilitating activation of both Golgi and synaptic fusion proteins ultimately converge onto N-ethylmaleimide (NEM)-sensitive factor. Activation of oxidation, PKA, and Epac pathways simultaneously cannot overcome inhibition of NSF by NEM treatment , confirm2+. Acid sphingomyelinase generates ceramide by cleavage of sphingomyelin, which promotes negative membrane curvature  vesicles (enlargeosomes). First identified in PC12 clones , enlarge2+ into the target cell.In addition to mechanically-induced membrane wounds, the plasmalemma can be disrupted by the insertion of stabilized membrane pores. Bacterial toxins such as streptolysin O damage cells through the insertion of large transmembrane pores that induce cell death and/or nutrient release  have been proposed to explain the initial seal  Dense packing of vesicles at the site of injury creates a longer and narrower diffusion path, possibly involving vesicle to vesicle attachment proteins such as gap junctions, adherens, and desmosomes :b) Contraction of an actomyosin ring See  surroundc) Internalization or budding of membrane, mediated by ESCRT. Endocytosis could remove and subsequently seal small (<100 nm) plasmalemmal disruptions .d) Fusion of vesicles to form larger or even multi-layered vesicles that would continue to accumulate at the wound site. In crayfish MGAs, squid GAs, and earthworm GAs, plasmalemmal injury induces formation of single-walled vesicles of plasmalemmal origin and multi-walled vesicles likely of glial origin .In contrast to a single-step patch seal by a wound vesicle, the vesicle plug hypothesis assumes that plasmalemmal holes or complete transections of cytoplasmic extensions are sealed by an accumulation of vesicles which more gradually forms a diffusion barrier through vesicle-vesicle and vesicle-plasmalemmal interactions. Vesicles and other membrane bound structures  interact with each other and the plasmalemma at the lesion site to form a “vesicular plug,” that form a diffusion barrier to particles of smaller and smaller molecular size. Several biophysical/morphological mechanisms likely play a role in the formation of a vesicle plug, including:a. Morphological observations show a gradual accumulation of membranous structures at sites of disruption associated with a diffusion barrier to smaller and smaller molecules  rather tXenopus oocytes and unfertilized sea urchin eggs . Modified SEM techniques that eliminate artifacts due to oxidation during fixation  could diXenopus oocytes , increased PKC activity, and increased cytosolic cAMP concentrations  vesiculation while subsequent wounding at a different site yields global vesiculation throughout the cell . The iniIn addition to the short-term adaptive resealing responses in wounded cells, long-term adaptations occur in both wounded and neighboring cultured MDCK cells. Both long- and short-term responses involve the phosphorylation of CRE-binding protein (CREB) that alters transcription. In 3T3 fibroblasts, membrane disruption stimulates PKC cascades. PKC then activates a p38 MAPK pathway that ultimately leads to CRE-mediated gene expression . PKC andLong-term adaptive responses are also observed in neighboring cells. Diffusible signaling molecules such as nitric oxide (NO) are released from wounded cells . NO/PKG Although relatively few adaptive membrane repair responses have been studied, the adaptive pathways and substances involved are similar to those reported for the repair of single plasmalemmal lesions. The biochemical pathways reported have not yet been correlated with observations of sub-cellular biophysical/morphological events, as is also the case for many single cell traumatic studies reported in previous sections.in vitro or in vivo. Solutions of 1–5 kDa PEG have been used for decades to generate cell hybrids in vitro . PEG of low molecular weight (0.2–7 kDa) causes an osmotic imbalance across the plasmalemma that may alter membrane curvature and induce mechanical stress . Howeverin vitro  and to rin vivo) of mammalian PNS axons brought into contact by neurorrhaphy and immediately restores involuntary muscle contractions. PEG-fused nerves exhibit return of voluntary behaviors after 2–6 weeks by inducing rewiring of PNS and CNS synaptic connections , perhaps by sealing off cut ends . In this model, application of PEG to the proximal or distal stump of a transected axon causes the rapid collapse and fusion of the axolemmal leaflets, similar to the previously assumed mechanism of axonal repair depicted in 2+ through the lesion site -- and perhaps secondarily by release of Ca2+ from intracellular structures . These parallel biochemical pathways initiate less well-defined cellular events that facilitate membrane fusion and repair.Eukaryotic cells commonly experience plasmalemmal damage. For decades prior to 1994, the cellular/molecular mechanisms that repair plasmalemmal injuries were not discussed or were misinterpreted. Such undocumented theories of membrane repair included lipid spreading, plasmalemmal collapse and fusion, and the formation of membrane partitions. Since 1994, experimental studies consistently show that small or large plasmalemmal lesions seal by accumulation and aggregation of vesicles and/or other membranous structures. A combination of electrophysiological measures, fluorescent dye with series confocal Z-sections, and high-resolution EM images of the injury site are necessary to accurately determine the morphological and functional state of a plasmalemmal seal. Vesicles and membrane-bound structures interact with the plasmalemma and each other to produce a complete seal by forming (1) a vesicular plug consisting of many vesicles that gradually seal the membrane disruption or (2) a patch consisting of a large wound vesicle that seals the membrane disruption in a single step. Both plug and patch sealing mechanisms assume that a set of parallel biochemical pathways and substances are primarily activated by the rapid influx of CaAlthough many publications cited above provide detail on the biochemical pathways of plasmalemmal repair, few studies correlate subcellular morphological/biophysical events directly associated with biochemical substances. For instance, very little is known regarding the transport of wound-induced vesicles in cells. A “tether” that anchors vesicles to the plasmalemma has been described in crayfish MGAs  perhaps 2+-dependent manner . However, it was only when high-resolution fluorescence imaging was performed on mouse perivascular cell culture that the cellular effect of AnxA5 was discovered. AnxA5 associates with PS-rich domains that surround the site of injury, forming rigid 2D crystals of AnxA5 trimers. AnxA5 arrays act to prevent expansion of the membrane injury and facilitate repair  in a Cae repair . This diA better understanding of both natural mechanisms of plasmalemmal repair are almost-certainly critical to the development of new clinical treatments for conditions that involve damage to nerve cell membranes such as traumatic nerve injury, neurodegenerative diseases, muscular dystrophies, stroke and other ischemic conditions. Many neurodegenerative diseases are characterized by damage to cell membranes  the membrane dynamics , 2) the presence or absence of intracellular vesicles, and 3) the assessment of a seal by dye exclusion. These properties will allow for study of the cellular dynamics upon PEG addition. Additionally, many of the inhibitors of substances within the natural repair biochemical pathway have been used previously in crayfish MGAs see  allowingvia local application of PEG could prevent apoptosis and improve behavioral recovery.PEG-sealing of may provide new avenues for the treatment of locally damaged cells and aid in the improvement of behavioral recovery following PEG-fusion of peripheral nerve injuries. Ischemic conditions  that are characterized by oxygen glucose deprivation in the cell often result in plasmalemmal damage and the formation of membrane blebs (


Idiopathic intracranial hypertension is a disease characterized by increased intracranial cerebrospinal fluid volume and pressure without evidence of other intracranial pathology. Dural sinuses are rigid structures representing a privileged low-pressure intracranial compartment. Rigidity of dural sinus ensures that the large physiologic fluctuations of cerebrospinal fluid pressure associated with postural changes or to Valsalva effect cannot be transmitted to the sinus. An abnormal dural sinus collapsibility, especially when associated with various anatomical sinus narrowing, has been proposed as a key factor in the pathogenesis of idiopathic intracranial hypertension. This pathogenetic model is based on an excessive collapsibility of the dural sinuses that leads to the triggering of a self-limiting venous collapse positive feedback-loop between the cerebrospinal fluid pressure, that compresses the sinus, and the increased dural sinus pressure upstream, that reduces the cerebrospinal fluid reabsorption rate, increasing cerebrospinal fluid volume and pressure at the expense of intracranial compliance and promoting further sinus compression. Intracranial compliance is the ability of the craniospinal space to accept small volumetric increases of one of its compartments without appreciable intracranial pressure rise. In idiopathic intracranial hypertension, a condition associated with a reduced rate of CSF reabsorption leading to its volumetric expansion, a pathologically reduced IC precedes and accompanies the rise of ICP. Syncope is defined as a transient loss of consciousness due to a transient cerebral hypoperfusion characterized by rapid onset, short duration, and spontaneous complete recovery. A transient global cerebral hypoperfusion represents the final mechanism of syncope determined by cardiac output and/or total peripheral resistance decrease. There are many causes determining low cardiac output including reflex bradycardia, arrhythmias, cardiac structural disease, inadequate venous return, and chronotropic and inotropic incompetence. Typically, syncopal transient loss of consciousness is mainly referred to an extracranial mechanism triggering a decrease in cardiac output and/or total peripheral resistance. Conversely, the association of syncope with a deranged control of intracranial compliance related to cerebral venous outflow disorders has been only anecdotally reported.We report on a 57-year-old woman with daily recurrent orthostatic hypotension syncope and idiopathic intracranial hypertension-related headaches, which resolved after lumbar puncture with cerebrospinal fluid subtraction.A novel mechanism underlying the triggering of orthostatic syncope in the presence of intracranial hypertension-dependent reduced intracranial compliance is discussed. Idiopathic intracranial hypertension (IIH) is a disease characterized by increased intracranial volume and pressure (ICP) without evidence of other intracranial pathology. Current IIH population prevalence is 2–22/100,000 . An incr2O [Dural sinuses are rigid structures representing a privileged low-pressure intracranial compartment. Rigidity of dural sinus ensures that the large physiologic fluctuations of CSF pressure associated with postural changes or to Valsalva effect  cannot 2O . This pa2O  stabiliz2O .Syncope is defined as a transient loss of consciousness (TLOC) due to a transient cerebral hypoperfusion characterized by rapid onset, short duration, and spontaneous complete recovery . The incTypically, syncopal-TLOC is mainly referred to an extracranial mechanism triggering a decrease in cardiac output and/or total peripheral resistance. Anecdotic evidence of syncope associated with significant intracranial pressure increase consequent to cerebral venous thrombosis have been reported , 12. We A 57-year-old overweight woman presented with long-standing history of syncopal TLOC, which began in adolescence as sporadic (every 3–4 months) but worsened over time (up to daily). Most syncopal events occurred in standing position anticipated only by brief prodromal symptoms which included nausea, sweating and pallor. Occasionally she reported urinary incontinence related episodes. She also suffered from migraine-like headache attacks which began at the age of 23 increasing in frequency over time up to a chronic pattern (> 20 headache days per month). Headache worsened in the lying position and could present at night, awakening the patient early in the morning. Tinnitus, dizziness, sporadic vertigo attacks and occasional transient diplopia were also reported. Syncopal events were mostly independent from the presence or intensity of migraine-like pain.2O, dropping to 8 cmH2O after 42 ml of CSF subtraction. Therefore, a diagnosis of IIHWOP was made accordingly to the Modified Dandy Criteria [Neurological examination was normal. Ocular fundoscopy was performed by an expert ophthalmologist, with no evidence of papilledema. Cardiac causes of syncope were excluded after extensive cardiological evaluation. Supine and standing blood pressure measurements did not show orthostatic hypotension and carotid sinus massage was negative. Tilt test was executed at admission, reporting a VASIS 2b response (cardioinhibitory response with asystole of more than three seconds) and therefore the patient was proposed for a pacemaker implantation. MR Venography (MRV) was performed, showing bilateral sinus stenosis, and, precisely, a transverse and sigmoid sinus hypoplasia on the right and two apparent flow gaps on the left at the junctions between Superior Sagittal Sinus (SSS) and left Transverse Sinus (TS) and between left TS and left Sigmoid Sinus. Apparent flow gaps are linked to a segmental flow velocity increase by focal sinus compression . Brain MCriteria , 15. AftFollow-up visits during the first year confirmed the disappearance of both syncopal episodes and chronic headache. Then symptoms progressively recurred with frequent migraine-like headaches and pre-syncopal or syncopal events. Again, syncopal events were independent from presence or intensity of migraine-like pain.2O OP, dropping to 10.2 cmH2O after 36 ml CSF withdrawal. On the following day, the patient reported on the resolution of the headache and a new tilt test confirmed VASIS 1 response. At two and six-months follow-up the patient reported the absence of syncopal episodes and only sporadic migraine-like attacks.A second LP, performed after 18 months, showed 20.3 cmHWe described a case of long-standing history of recurrent syncope in the presence of sinus stenosis associated IIHWOP, which showed sustained remission after LP with CSF subtraction. After symptoms relapse, a new LP showed again good prompt response, thus suggesting a direct link between the increased intracranial pressure and the recurrent syncope. In recent years, IIH have been found in about 9% of a cohort of patients with orthostatic intolerance . Interesvolumetric expansion, a pathologically reduced IC precedes and accompanies the rise of ICP. A reduced IC amplifies the physiological fluctuations of CSF pressure: large “beta waves” encompassing the upper limit of the normal ICP range promptly normalize after CSF subtraction or after medical treatment [The intracranial compliance (IC) is the ability of the craniospinal space to accept small volumetric increases of one of its compartments without appreciable intracranial pressure (ICP) increase. Formally it is defined as the change in CSF volume per unit of change in CSF pressure . After areatment . There ireatment .Mechanisms underlying IC are not fully understood. A low-grade collapsibility of the dural sinuses is probably physiological and may contribute to IC generation by displacing some venous blood outside the cranial space. Another and probably more relevant mechanism is the displacement of CSF through the foramen magnum towards the spinal space, up to the dural sac. Indeed, the dural sac volume increases in condition of increased CSF volume and pressure, and decreases under the Valsalva manoeuvre, which by contrast raises volume and pressure of the epidural venous plexus surrounding the sac . Thus, tIC also influences the cerebral perfusion dynamics. In subjects with preserved IC, during the transition to orthostatic position, a displacement of a small amount of intracranial CSF towards the spinal space occurs physiologically. The displaced CSF volume reduces the intracranial pressure and, consequently, also the cortical vein pressure which is dynamically coupled with ICP due to the Starling resistor properties of the bridging vein  . Thus, dvolume and pressure, preserving the IC.In subjects with increased CSF volume and pressure, by definition, the IC is lacking or very low. During the transition to orthostatic position, the expanded CSF volume prevents any CSF dislocation towards the spinal cavity. Consequently, the ICP and the strictly ICP-coupled cortical vein pressure, do not decrease parallel to the arterial pressure reduction under gravity effect. This pressure mismatch temporarily reduces the CPP up to induce a syncope, likely also boosting the vasovagal reflex. Stok et al.  providedpredisposed individuals. The nature of such predisposition is unknown but possibly linked to a delayed compensatory cardiovascular reflex response.In summary, our case suggests that even when relatively isolated—meaning not associated with definitely pathological OP – a compromised IC hinders the displacement of a portion of cerebrospinal fluid (CSF) into the spinal canal during the transition to an orthostatic state, preventing the drop of intracranial pressure . Since tIntracranial compliance plays a fundamental role in cerebral perfusion homeostasis. An intact IC is required to ensure the physiologic rearrangement of brain perfusion dynamics in course of postural changes. In subjects with sinus stenosis dependent rise in dural sinus pressure, the reduced CSF reabsorption expands the CSF volume pathologically reducing the IC. This mechanism precedes the onset of a detectable increased intracranial pressure and may be isolated in some individuals, i.e. not associated with a defined CSF pressure increase. In the transition to orthostatic position, a reduced IC prevents the gravity–dependent cortical venous pressure drop while the arterial pressure physiologically decreases by gravity. The mismatch leads to a transient acute fall of CPP, eventually inducing a syncope. Considering the possible high prevalence of asymptomatic forms of sinus stenosis-associated IIHWOP , a patho


MOMA successfully predicts the overall survival, disease-free survival , and copy number alterations of CRC patients. In addition, our approaches identify interpretable pathology patterns predictive of gene expression profiles, microsatellite instability status, and clinically actionable genetic alterations. We show that MOMA models are generalizable to multiple patient populations with different demographic compositions and pathology images collected from distinctive digitization methods. Our machine learning approaches provide clinically actionable predictions that could inform treatments for colorectal cancer patients.Histopathologic assessment is indispensable for diagnosing colorectal cancer (CRC). However, manual evaluation of the diseased tissues under the microscope cannot reliably inform patient prognosis or genomic variations crucial for treatment selections. To address these challenges, we develop the Multi-omics Multi-cohort Assessment (MOMA) platform, an explainable machine learning approach, to systematically identify and interpret the relationship between patients’ histologic patterns, multi-omics, and clinical profiles in three large patient cohorts ( Histopathological analysis is an essential tool in diagnosing colorectal cancer, but is limited in predicting prognosis and molecular profiles. Here, the authors designed a machine learning-based platform to predict multi-omics profiles and prognosis from pathology images. Histopathologic evaluation remains a cornerstone for diagnosing and staging CRC, and the histology subtypes and genetic variations are the keys to treatment selection2. However, inter-rater variability in histopathology diagnoses has been reported3, and the genomic profiling process requires days to weeks to complete and is not available to every hospital in the developing world. These limitations have hindered CRC patients from receiving timely and appropriate treatments.Colorectal cancer (CRC) is the second most common cause of cancer death in the United States, accounting for nearly 53,000 deaths annually4. Several studies using machine learning techniques reported remarkable diagnostic accuracy for various cancer types, such as lung, breast, ovarian, renal cell, and colorectal carcinomas11. Previous works also demonstrated unexpected correlations between histopathology image features and clinically actionable molecular variations, such as microsatellite instability and PTEN gene deletion, in colorectal carcinoma samples13. These studies indicate that high-resolution pathology images contain underutilized biomedical signals useful for personalizing cancer care19.With the recent development of reliable whole-slide pathology scanners and high-performing computer vision techniques, quantitative pathology evaluation has become increasingly feasible20. Typical high-resolution digital pathology whole-slide images of colorectal cancer tissues contain up to billions of pixels, making it infeasible for standard convolutional neural networks to process the whole image at once. In addition, deep learning models are highly complex, and it is difficult to connect the image patterns discovered by these data-driven models with biological knowledge21. Furthermore, since there are a large number of parameters that researchers need to optimize in data-driven machine learning models, generalizability to other image acquisition methods remains a substantial challenge to many digital pathology models22. The lack of extensive validation in different patient cohorts can diminish the applicability of machine learning models in clinical settings.Nonetheless, many computational challenges hinder the extraction of useful histopathology signals, and several reports expressed concerns about the generalizability of deep learning modelsIn this study, we propose the Multi-omics Multi-cohort Assessment (MOMA) system, an explainable machine learning framework for analyzing digital pathology images at scale. Our informatics methods successfully predict the prognoses of early-stage colorectal cancer patients and achieve state-of-the-art performance in identifying the genomics and proteomics status of cancer samples using a weakly supervised prediction framework. We connect high-resolution digital pathology images with clinically actionable multi-omics aberrations, and we identify interpretable pathology predictors of patients’ survival outcomes. We further validate our framework in multiple large patient cohorts and demonstrate its generalizability in different populations and using different image acquisition methods. Our study provides a robust and flexible machine learning framework for scalable histopathology image analyses.We develop the Multi-omics Multi-cohort Assessment (MOMA) machine learning framework for predicting clinically actionable variations in cancer genomics, proteomics, and patient prognoses using histopathology images. Figure P = 0.0495) and PLCO , demonstrating the generalizability of our approaches. We visualize our models and show that dense clusters of adenocarcinoma cells are highly indicative of worse overall survival outcomes . When stratifying the datasets into colon cancer and rectal cancer groups, our approaches successfully identify the prognostic differences in both groups  and PLCO . On model visualization, we show that dense clusters of adenocarcinoma cells are highly indicative of worse overall survival outcomes . Similar to our overall survival results, model visualization shows that dense clusters of adenocarcinoma cells are highly indicative of worse progression-free outcomes  status has been identified as a biomarker that predicts the response to immune checkpoint inhibitors. To facilitate the treatment effectiveness prediction for immune checkpoint inhibitors, we employ MOMA to predict the MSI status of each patient. Results show that the AUROC of the TCGA held-out test set is 0.88 ± 0.06 Fig. , and in BECN1 gene using histopathology images. CNAs of many key genes, including FHIT and PTEN, have been implicated in carcinogenesis. Here we show that MOMA predicts CNAs in FHIT and many other tumor suppressor genes , whole-genome doubling, and overexpression of the nes Fig. . Comparenes Fig. . In addiers Fig. . FurtherBECN1  mutation in the TCGA test set, with an AUROC of 0.71 ± 0.07  with those without BRAF mutation, we also show that MOMA can identify the morphological signals associated with BRAF mutations in general  colorectal cancer is a subtype characterized by widespread hypermethylation of promoter CpG islands. This hypermethylation pattern inactivates many tumor suppressor genes and causes global gene expression dysregulations and metabolic alterations. Previous studies suggest that patients with CIMP-high status have worse prognoses under the standard treatments25. To identify the histopathology patterns indicative of the CMS subtypes, we employ MOMA to classify the major CMS subtypes with sufficient numbers of samples (CMS2 and CMS4). Results show that MOMA achieved an AUROC of 0.66 ± 0.04 in the held-out test set not participating in the model development process  is a commonly used molecular subtyping system for colorectal cancer that addresses inconsistencies in gene-expression-based classifications and reflects the biological differences in tumor characteristicsWe summarize the regions indicative of patients’ prognostic outcomes and molecular profiles identified by our interpretable machine learning framework. Our approaches provide a quantitative measurement of the relative importance of each region in predicting these outcomes of interest. For example, we show that histological patterns of the tumor, stroma, and mucus regions are relevant to the prediction of overall survival and disease-free survival, while regions with lymphocytes and mucus provide signals for predicting CIMP-high status. Figure 26. Our methods can automatically identify informative regions from whole-slide pathology images without the need for detailed region-level annotations. In addition, we employed the vision transformer and obtained significantly improved performance compared with that of standard deep learning methods12. Our multi-cohort validation showed the generalizability of our data-driven approaches for analyzing high-resolution digital pathology images.In this study, we designed the MOMA framework for molecular characterization and clinical prognostic prediction using histopathology images of colorectal cancer, and we further validated our models in two independent patient cohorts. Our results demonstrate that interpretable machine learning approaches can predict patients’ survival outcomes and clinically important molecular profiles27.Our models demonstrated that high-resolution histopathology slides contain useful predictive signals for genetic aberrations and survival outcomes. Because genetic profiling requires additional tissue samples, processing time, and costs, our prediction models that use only the H&E-stained histopathology slides can provide timely decision support for treatment selection in resource-limiting settings or in clinical scenarios with limited tissue availability. In addition, our stage-stratified survival outcome prediction successfully identified patients with shorter overall and disease-free survival under the standard treatments. These results showed that our machine learning approaches extracted stage-independent morphological signals indicative of patients’ clinical outcomes. Because patient prognosis depends on many clinical factors, no prediction models can perfectly identify the survival outcomes of individual patients. Nonetheless, our approach unveiled histopathology patterns related to patient prognosis, which could be useful in guiding clinical decision-making. For example, clinicians may provide closer follow-up to patients with suboptimal clinical prognoses, consider more aggressive treatment options, or enroll them in ongoing clinical trials12 for MSI prediction using the TCGA dataset, and we showed that MOMA achieved a 4% improvement on the same dataset  status. The MSI-high status is an established biomarker for responses to immune checkpoint blockade39, while BECN1 is a key regulator of autophagy40 and has been proposed as a potential target for immunotherapy41. Our findings confirmed the relevance of these biomarkers with immune cell infiltration and suggested the role of digital pathology profiling in the prediction of response to immunotherapy.Due to the large number of parameters in deep learning models45, which may be useful for enhancing deep learning-based pathology feature extraction. Future research can investigate the benefits and potential caveats of these methods. Furthermore, incorporating patients’ radiology imaging data, pathology profiles, molecular aberrations, and clinical characteristics may further improve the prognostic prediction for colorectal cancer patients. Additional research is required to identify the optimal prognostic prediction methods and enable personalized treatments and advance care planning.Our study has a few limitations. First, our study is based on patient populations in North America. Although our results are validated in two large-scale, independent, and diverse patient cohorts, additional studies that focus on specific patient populations are needed to evaluate the applicability of our models in the targeted clinical settings. In addition, recent studies on self-supervised machine learning hinted at the potential for enhanced representation learning for efficient machine learningIn summary, we presented an interpretable machine-learning framework that systematically identifies the relationships between histopathology, molecular variations, and patients’ survival outcomes. We successfully predicted key genetic aberrations, gene expression profiles, overall survival, and progression-free survival in colorectal cancer patients, with the results validated in two independent validation cohorts. Our approaches can be extended to characterize the prognostic-informing quantitative pathology patterns of other complex diseases.46, the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO)47, the Nurses’ Health Study (NHS)48, and the Health Professionals Follow-up Study (HPFS)50. We acquired the digital whole-slide pathology images, whole-exome sequencing results, and RNA-sequencing data of TCGA patients from the National Cancer Institute Genomic Data Commons Portal . Mutation status, copy number alterations (including genetic amplifications and deletions), microsatellite instability, and CpG island methylator phenotypes (CIMP) of both colon and rectal adenocarcinomas were extracted from the cBioPortal . Whole genome doublings and consensus molecular subtypes (CMS) of colorectal cancers were obtained from a previous TCGA publication51.We obtained histopathology images of colon and rectal cancer patients from The Cancer Genome Atlas (TCGA) tissue slide dataset52. For each histopathology from the NHS and HPFS cohort, two experienced colorectal cancer pathologists reviewed the cancer samples and selected the cores to ensure the representativeness of the cores. Thus, the TMA images include regions of tumor cells, stroma, tumor/stromal interfaces , lymphocyte infiltration, and other pathological changes characteristic of the tumor sample from which the core was generated. Our multi-center study was approved by the Institutional Review Boards of Harvard Medical School (IRB20-1509). Our study protocol was also approved by the Brigham and Women’s Hospital, Harvard T.H. Chan School of Public Health, and the participating registries as required.In addition, we obtained PLCO data from the National Cancer Institute Cancer Data Access System, and we collected clinical, genomic profiles, immunohistochemistry, and hematoxylin-and-eosin (H&E) stained tissue microarray images from the NHS and the HPFS coordinated by Harvard T.H. Chan School of Public Health, Harvard Medical School, and Brigham and Women’s Hospital. Notably, colorectal tumor tissue blocks in the NHS and the HPFS were retrieved from over a hundred hospitals throughout the U.S. with variable tissue age, which increased the generalizability of our findings53 to account for the staining differences across tissues and convert pixel values to a similar space in optical density. We used convolutional neural networks and vision transformers to extract pathology image features from each tile and connect these features with genomics, gene and protein expression levels, as well as patients’ overall survival and disease-free survival outcomes.We designed a Multi-omics Multi-cohort Assessment (MOMA) machine learning framework to enable robust predictions of cancer genomics, proteomics, and important clinical outcomes. In this framework, we first pre-processed the whole-slide histopathology images by tiling each image into patches with 1000×1000 pixels, and we employed the color normalization proposed by Macenko et al.54, multi-modality outcome prediction frameworks32, and biological interpretations of the prediction models. Leveraging state-of-the-art vision transformer models, we extracted informative features from the whole-slide images, and we connected them with genomic mutations, copy number alterations, transcriptomic profiles, and survival outcomes by incorporating relevant statistical models  to predict the molecular profiles and clinical outcomes of each individual. We further provided biological interpretations of the model predictions using pathology concepts of the tumor microenvironment55. Below we describe our methodology in detail.One key feature of MOMA is the integration of multiple-instance learningBRAF c.1799T > A (p.V600E) mutation (48 patients with BRAF c.1799T > A (p.V600E) tumor; 529 patients with BRAF wild-type tumor), and the most prevalent Consensus Molecular Subtypes  patients; 105 CMS4  patients) of colorectal cancer. These tasks were constructed as weakly-supervised classification tasks.Using the MOMA platform, we conducted multi-omics subgroup predictions on colorectal cancer patients, with a focus on clinically actionable molecular aberrations. Specifically, we predicted the microsatellite instability (MSI) status , CpG island methylator phenotype , 56, to extract 2048 features from the image patches. To mitigate the impact of artifacts in the whole-slide images, we applied the k-means algorithm to cluster the extracted feature vectors into 10 clusters, because typical colorectal cancer pathology images contain 10 different types of regions with biological significance , and we used vision transformers57 to derive the informative features for each cluster. The clusters whose weights were in the top three (strong positive association) or bottom three (strong negative association) would be used in the downstream analyses. After the vision transformer, the dimension of the feature vectors of each cluster was reduced to 512 to obtain efficient image representations for multiple-instance learning60. We used a trainable attention-based pooling operation to aggregate these feature vectors. Our transformer encoder layers contain 512 neurons in the hidden layer, 8 heads, and 2048 neurons in the multi-layer perceptron, with a dropout rate of 0.1.We employed the ResNet-50 network, a residual neural network with 50 layers61 with varying hyperparameter tau optimized for each task. We computed the total loss of the model as the sum of the bag loss function and the instance loss function.Finally, we applied two loss functions in the prediction tasks. The first one was the bag loss function of standard binary or multiclass cross-entropy with the inverted class weights informed by the number of tiles in each class. The inverted class weights enabled machine learning models to account for the classes with fewer instances and prevent the models from biasing toward predicting all instances as the majority class. The other was the instance loss function of the tile-level classifiers. To compute the instance loss function, we first ranked the weights obtained from the attention-based multiple-instance learning to select the top three clusters with positive labels and the bottom three clusters with negative labels. Next, we employed the smooth support vector machinehttps://rebrand.ly/MOMA_demo) that allows users to upload pathology images and employ our trained models to generate predictions. Our source codes for data analyses and trained models could be found at https://github.com/hms-dbmi/MOMA.To develop our models, we first split the TCGA dataset into 60% training, 20% validation, and 20% test sets. All tiles from the same whole-slide images were put in the same partition, in order to prevent information leaks. We trained our models using the training set, selected the optimal hyperparameters using the validation set, and reported our results in the untouched test set. We further validated our models in independent validation cohorts . We reported the area under the receiver operating characteristic curves (AUROCs) of the test set for each classification task. We used a stochastic gradient descent (SGD) optimizer with a learning rate of 1e-3, a momentum of 0.9, a batch size of 1, and a weight decay of 5e-4. We trained all models with 250 epochs with a cosine annealing learning rate scheduler. We implemented our methods using Python 3.6 with PyTorch 1.6.0 in a single GPU system with NVIDIA Titan RTX. To make MOMA easily accessible to pathologists, oncologists, and biomedical informatics researchers, we further developed a web portal  and stage III colorectal cancer patients. We distinguished patients in the same stage groups into a “predicted longer-term survival group” and a “predicted shorter-term survival group,” and we used the log-rank test to evaluate their differences in actual survival outcomes. Stage IV patients received heterogeneous treatments and were thus not included in our stratified survival outcome prediction analyses. The Weibull distribution is a probability distribution with shape (kappa) and scale (lambda) parameters. Combinations of the shape and scale parameters can model different hazard functions for survival analyses. We modified our machine learning framework to estimate these two statistical parameters in the Weibull survival model. We used a trainable attention-based pooling operation to aggregate the image feature vectors and employed the exponential activation function for lambda and softplus for kappa63. Our deep learning-based Weibull modeling approach can handle right censoring and accommodate different patterns of death rate over time . Due to the smaller sample size in the TCGA dataset for the survival prediction task (337 patients with stage I or II cancer and 181 stage III patients), we first conducted a 5-fold cross-validation on TCGA before validating our approaches in external validation cohorts. We divided the prediction results into shorter-term survival and longer-term survival groups using the median predicted survival index, and we tested the survival differences between the predicted groups using the log-rank test. We used an RMSprop optimizer with a learning rate of 1e-5 and trained the model with 5 epochs using a batch size of 1. We implement our training and testing processing using Python 3.6 with TensorFlow 2 in the same single GPU system.To demonstrate the extensibility of our MOMA platform to different prediction tasks, we connected our machine learning framework with the Weibull modeling methodsp-value  in these independent validations.To investigate the generalizability of our machine learning models, we harnessed two additional cohorts collected at different hospitals. Specifically, we used the pathology tissue microarray, genetic, immunohistochemistry, and clinical datasets from the NHS and HPFS to validate our trained prediction models. We further validated our survival prediction models using the whole-slide histopathology and survival information from the PLCO cohort. We applied the same image tiling, pre-processing, and color normalization methods to preprocess histopathology images from these external cohorts. We reported the AUROC (for classification tasks), concordance index (c-index), and log-rank test We further identified human-interpretable pathology features employed by our machine learning models to obtain biological insights into the connections between histopathology morphology and molecular profiles. We developed a model interpretation method that incorporates model-derived concept scores and expert-annotated concepts based on prior pathology knowledge. Specifically, we first quantified the importance of each image region by occluding all pixels in the region and computing the extent to which the predicted outcome changed when the region was occluded. We define the importance index of each image region as the numerical change of the predicted probability due to the occlusion of the region. To connect crucial regions with pathology interpretation, we leverage 100,000 histopathology images annotated by gastrointestinal pathologists with seven concepts: colorectal adenocarcinoma epithelium, cancer-associated stroma, lymphocytes, smooth muscle, mucus, adipose tissue, and tissue debris. We developed a deep learning model that classified image regions into these pathology concepts with an accuracy of 99.38%, and we employed this model to compute the concept scores for regions with importance indices greater than 0.7. We scaled our concept scores to a range of , where 100 indicates the region has the highest relevance to the concept of interest. Thus, our concept scores indicate the amount of attention our machine learning model pays to different regions of pathology changes in making the prediction, and it is not directly related to the amount of area occupied by each pathology pattern in the slides. We repeated this process for each machine learning task we performed. We used the standard color map to visualize the importance index and overlaid it with the original histopathology images. These approaches provide intuitive model interpretations using well-established concepts in cancer pathology.No data were excluded from the analyses. All available samples were included in the machine learning analyses, and the experiments were not randomized. The investigators were blinded to the labels of the samples in the test set before the final model evaluation.Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1


Transtendon repair and repair after completion of the tear have been widely used to treat partial-thickness rotator cuff tears (PT-RCTs). The present study was aimed to compare the clinical outcomes and tendon integrity following arthroscopic repair of articular PT-RCTs using transtendon repair or repair after completion of the tear.We performed a systematic electronic database search on Cochrane Central Register of Controlled Trials, PubMed and Embase to identify articles equating articular-sided PT-RCTs repair. The randomized controlled clinical trials that met our criteria were evaluated for quality of methodology. The results obtained were further analyzed and correlated to present the benefits and drawbacks comparing the two surgical procedures.p > 0.05).According to our inclusion and exclusion criteria, six articles were included in the present study. A total of 501 patients were analyzed as part of this study. The results indicated that both the surgical treatments provided excellent functional improvements and tendon integrity. However, no significant differences for the visual analogue scale (VAS) score, American Shoulder and Elbow Surgeons (ASES) score, constant score, range of motion, postoperative adhesive capsulitis, tendon integrity and patient satisfaction were found between the two cohorts (Both transtendon technique and repair after completion of the tear for articular-sided partial rotator cuff tear provide improvements in clinical outcome with a low complication rate and a high rate of healing. Partial rotator cuff tears (PT-RCTs) are a common cause of shoulder pain and disability and can be classified as bursal side, articular side, or intratendinous in accordance with the injury site . Ellman Generally, surgical alternatives for treating symptomatic articular-sided PT-RCTs include arthroscopic repair following full-thickness conversion and various in situ repairs  Some surThe purpose of this study was to compare functional outcomes and tendon integrity for patients with an articular-sided PT-RCTs who undergone arthroscopic transtendon repair or tear completion repair. We hypothesized that arthroscopic repair of articular-sided PT-RCTs would provide satisfactory functional improvements and tendon integrity regardless of the two repair technique.We carried out a meta-analysis of the literature with the search terms “partial”, “articular” and “rotator cuff tear”. A complete search of the literature in the following databases was performed: MEDLINE (PubMed) (1950 to June 2021), Embase (Ovid) (1974 to June 2021) and Cochrane (1996 to June 2021). Our inclusion criteria included outcome-based studies of isolated partial articular-sided rotator cuff tears (> 50% of the thickness of the tendon) which compared arthroscopic transtendon repair and tear completion repair by using clinical or functional scoring systems. Exclusion criteria included studies that involved cadaver or animal studies, biomechanical studies, literature reviews, letters to editors, expert opinion articles, case reports or technique notes which did not contain clinical outcome-based data.The Coleman methodology score (CMS) was applied to determine the quality of the involved studies according to our previous study . The CMSThe identified studies were measured and analyzed for the following outcomes: visual analogue scale (VAS) score, American Shoulder and Elbow Surgeons (ASES) score, Constant score, range of motion, postoperative adhesive capsulitis, tendon integrity and patient satisfaction. Two reviewers evaluated the literature separately, and any discrepancies were reevaluated and resolved by consensus.A formal meta-analysis was conducted only for clinical outcome data from comparative studies using Stata 12.0 . Results for continuous or categorical outcomes were reported as a mean difference or an odds ratio, respectively, with 95% confidence intervals.n = 576), Embase (n = 570) and Cochrane (n = 149). Six articles that met our inclusion and exclusion criteria were subsequently included after a full-text review . No significant difference was observed regarding the VAS score between the two cohorts P = 0.525) . No significant difference was observed between the two cohorts for the ASES score (P = 0.515) , implying that no significant difference was found between the two cohorts P = 0.463)   while the standard mean difference of external rotation was -0.254 (P = 0.190). No significant difference was found between the two cohorts  was calculated with a P value of 0.903, implying no significant difference in tendon integrity between the two groups (Fig. The main finding of our study is that both procedures provide improvements in clinical outcome with a low complication rate and a high rate of healing. No significant difference in clinical outcome was demonstrated between these two procedures for articular-sided partial rotator cuff tear repair.Articular PT-RCTs with a thickness of greater than 50% can be repaired by tear completion repair and the in situ transtendon procedure. Tear completion repair is a relatively straightforward method for restoration of the tendon footprint with satisfactory clinical outcomes and high levels of patient satisfaction. Some authors advocated tear completion repair because it allowed better access to the tendon footprint and tended to be more convenient to secure tendon fixation . FurtherIn contrast, the transtendon repair made it possible to better restore the rotator cuff footprint anatomically and maintain the tendon integrity as preserving the bursal side rotator cuff tendon . CadaverThe limitations of this study include a relatively small number of studies. Despite the small sample size, we included only level- II studies. Hence, these studies represent high-level evidence on a large number of patients. The preliminary results presented in this study would help to clarify clinical outcomes of both repair techniques. In addition, the heterogeneity of imaging modality may represent a weakness of the study. There is a lack of standardized evaluation method for accurate assessment of tendon thickness. According to a previous report, ultrasound and MRI have good and similar diagnostic accuracy for detection of rotator cuff tears, and they have been widely performed to evaluate rotator cuff integrity .Arthroscopic repair of partial-thickness articular-sided rotator cuff tears provided functional improvements and tendon integrity regardless of the transtendon repair and repair after completion of the tear. Due to the simple operation and the removal of an obvious degeneration of the rotator cuff, the procedure of repair after completion of the tear is proposed in the case of obvious degeneration of rotator cuff.