Conflicting results regarding alterations to sperm DNA methylation in cases of spermatogenesis defects, male infertility and poor developmental outcomes have been reported in humans. Bulls used for artificial insemination represent a relevant model in this field, as the broad dissemination of bull semen considerably alleviates confounding factors and enables the precise assessment of male fertility. This study was therefore designed to assess the potential for sperm DNA methylation to predict bull fertility.n = 57) or subfertile (n = 43) based on non-return rates 56 days after insemination. The DNA methylation profiles of these samples were obtained using reduced representation bisulfite sequencing. After excluding putative sequence polymorphisms, 490 fertility-related differentially methylated cytosines (DMCs) were identified, most of which were hypermethylated in subfertile bulls. Interestingly, 46 genes targeted by DMCs are involved in embryonic and fetal development, sperm function and maturation, or have been related to fertility in genome-wide association studies; five of these were further analyzed by pyrosequencing. In order to evaluate the prognostic value of fertility-related DMCs, the sperm samples were split between training (n = 67) and testing (n = 33) sets. Using a Random Forest approach, a predictive model was built from the methylation values obtained on the training set. The predictive accuracy of this model was 72% on the testing set and 72% on individual ejaculates collected from an independent cohort of 20 bulls.A unique collection of 100 sperm samples was constituted by pooling 2–5 ejaculates per bull from 100 Montbéliarde bulls of comparable ages, assessed as fertile (This study, conducted on the largest set of bull sperm samples so far examined in epigenetic analyses, demonstrated that the sperm methylome is a valuable source of male fertility biomarkers. The next challenge is to combine these results with other data on the same sperm samples in order to improve the quality of the model and better understand the interplay between DNA methylation and other molecular features in the regulation of fertility. This research may have potential applications in human medicine, where infertility affects the interaction between a male and a female, thus making it difficult to isolate the male factor.The online version contains supplementary material available at 10.1186/s13148-022-01275-x. Understanding the causes of infertility and subfertility is a challenge in human medicine as these conditions affect approximately 15% of couples. Infertility is defined as an inability to conceive a child after 12 months of unprotected intercourse. In 30% of cases, male factors are recognized as the primary cause of infertility because anatomical, hormonal or known genetic anomalies have been clearly diagnosed but no female factors have been identified , meaningMature spermatozoa represent the ultimate form of male germ cell differentiation, which is a tightly regulated process that starts in utero and is achieved in adulthood during spermatogenesis. Sperm cells are meant to survive outside the organism, fertilize an oocyte and contribute to a new individual; and although they are transcriptionally inactive, they bear a remarkable epigenome in line with their high degree of specialization and unique nature . Thus DNNumerous studies in human cohorts have identified specific DNA methylation profiles associated with altered semen parameters –25, embrUnlike the situation in humans, the widespread use of artificial insemination (AI) in dairy cattle and the dissemination of bull semen to many herds markedly reduces these limitations; the bull is therefore an excellent animal model to investigate the etiology of male subfertility. Indeed, each bull is usually mated with hundreds of cows maintained in different herds, and the outcome of each insemination is recorded. Bull fertility can therefore be measured accurately in the field and corrected for many confounding factors. Moreover, bull fertility is an important economic trait. Unsuccessful AI can indeed give rise to direct costs, extended calving intervals and increased culling rates of the inseminated cows. Considerable efforts have therefore been made in an attempt to predict bull fertility and to understand the causes of subfertility based on genotypes, semen functional parameters or sperm molecular features, including epigenetic modifications , 34. SomIn order to contribute knowledge in this field, we investigated the nucleotide-level resolution, genome-wide DNA methylation profiles of semen samples collected from a total of 120 carefully selected Montbéliarde bulls with contrasting fertility levels. We report here on the biological characterization of the loci that were differentially methylated between fertile and subfertile bulls, and on the potential for using these loci to build models predictive of fertility status.n = 57) or subfertile (n = 43) according to hundreds of AI records  were subjected to differential analyses using two types of algorithms see “”. While To suppress the polymorphisms affecting CpGs that could interfere with DMC detection, all putative variants recorded in the 1000 Bull Genome database and targeting the CpGs covered by RRBS were filtered out, resulting in a background of 1,548,563 CpGs10 covered in at least 22 samples per fertility group . Using methylKit, 490 DMCs and 46 differentially methylated regions (DMRs) were identified, which contrasts with no DMCs being obtained with DSS, and suggests that sources of intra-group inter-individual variations still existed in our dataset. This was also visible from the hierarchical clustering run on the CpGs10 before and after the filtering of sequence variants, which demonstrates that inter-individual epigenetic variations unrelated to fertility shape the sperm methylome independently of the presence of polymorphisms  and hypomethylated (38% vs. 16%) DMCs. However, at least for LINEs in the L1 family, a trend toward hypomethylation in subfertile bulls was observed using a Repbase genome that has the potential to capture information from the multimapped reads.PLXNA4 (chromosome 4), PLXNB2 (chromosome 5) and PLXNA1 (chromosome 22), which belong to the Plexin family, were associated with all the terms in the cluster. Plexins are present on the surface of cells and interact with the semaphorin family of proteins to trigger a rearrangement of the cytoskeleton and to regulate cell shape, differentiation, junctions, motility and survival. All these processes are involved in the remodeling of epithelia and endothelia during organogenesis as well as in axon guidance during development of the nervous system 26], 3 2 and 384 Additional file 1: Fig. S1.. correlation clustering run on the methylation percentages at CpGs10 covered in at least 22 samples per group. Fig. S2: heatmap run on CpGs discriminant between sperm and somatic cells. Fig. S3: annotation of fertility-related DMRs relative to different genome features. Fig. S4: average DNA methylation at individual LINE and LTR repeats after alignment of the RRBS sequences on a Repbase artificial genome. Fig. S5: pyrosequencing validation of the upstream region of LBX1 gene. Fig. S6: fertility, semen functional parameters and semen sample characteristics in correctly classified and misclassified bulls. Fig. S7: PCA run on the methylation percentages at DMCs, with or without the imputation of missing values. Fig. S8: semen functional parameters before and after correction for the batch effect.Additional file 2: Table S1. Listing the semen samples, information on their origin and processing, and the field fertility of the corresponding bulls.Additional file 3: Table S2. Listing the functional parameters measured on each semen sample.Additional file 4: Table S3. Listing the fertility-related DMCs, their DNA methylation status in each sample and their annotation regarding genome features.Additional file 5: Table S4. Listing the fertility-related DMRs, their methylation status in each fertility group and their annotation regarding genome features.Additional file 6: Table S5. Listing the CpGs10 obtained after alignment of the RRBS sequences on a Repbase artificial genome, their coverage and DNA methylation status in each sample.Additional file 7: Table S6. Listing the differentially methylated genes relevant to fertility together with supporting references.Additional file 8: Table S7. Listing the differentially methylated genes found in common with four human studies.Additional file 9: Table S8. Listing the semen samples used for pyrosequencing validation.Additional file 10: Table S9. Listing the primers used for pyrosequencing validation.


Plasma LCN2 and MOG-IgG levels were significantly correlated in patients with optic neuritis . There were no significant differences in the areas under the ROC curve (AUC) of plasma LCN2  and MOG-IgG  levels . Plasma LCN2 levels may aid differentiation of MOG-IgG–positive optic neuritis from MOG-IgG–negative optic neuritis.This study aimed to evaluate the correlation between plasma lipocalin-2 (LCN2) levels and myelin oligodendrocyte glycoprotein (MOG)-immunoglobulin G (IgG) seropositivity in patients with optic neuritis. Peripheral blood samples were collected from 19 patients with optic neuritis and 20 healthy controls. Plasma LCN2 and MOG-IgG levels were measured using enzyme-linked immunosorbent assay and a cell-based assay, respectively. The correlation between plasma LCN2 levels and MOG-IgG titers in patients with optic neuritis was analyzed. Receiver operating characteristic (ROC) curves were constructed to assess and compare the ability of plasma LCN2 and MOG-IgG levels for predicting optic neuritis recurrence. Patients with MOG-IgG–positive optic neuritis had significantly higher mean plasma LCN2 levels than controls and patients with MOG-IgG–negative optic neuritis ( Discovery of a novel glial autoantibody specific for myelin oligodendrocyte glycoprotein (MOG) has enabled recognition of antigen-specific inflammatory demyelinating diseases of the central nervous system (CNS) manifesting as optic neuritis ,2,3. MOGMOG-IgG–positive optic neuritis is a recently discovered cause of optic neuropathy. Its pathophysiological and clinical characteristics differ from those of optic neuritis associated with multiple sclerosis or neuromyelitis optica . PatientWe previously reported that lipocalin-2 (LCN2) expression was highly upregulated in the optic nerve after EAON induction through MOG immunization . LCN2, aTherefore, we hypothesized that measuring the plasma LCN2 levels in patients with optic neuritis may help in the diagnosis of MOG-IgG–positive optic neuritis. We believe that the correlation of plasma LCN2 levels with MOG-IgG titers in patients with demyelinating optic neuritis has not been previously reported. The purpose of this study was to measure plasma LCN2 levels and evaluate their correlation with MOG-IgG titers in patients with optic neuritis.This study was conducted from March 2018 to December 2020 at the Department of Ophthalmology, Kyungpook National University Hospital. The study was approved by the Institutional Review Board of Kyungpook National University Hospital and was conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from all subjects. Out of 28 consecutive patients with demyelinating optic neuritis identified, 19 were included in this study. Patients with multiple sclerosis, neuromyelitis optica, or symptomatic CNS lesions other than those of the optic nerve; patients with incomplete clinical data; and patients who were followed up for less than 1 year were excluded. All patients had no history of neurological disease or new neurologic symptoms associated with their visual disturbance. As controls, we included 20 age- and sex-matched healthy individuals who had no conditions that could affect plasma levels of LCN2, such as infection, inflammatory disease, diabetes, or cancer, and had best-corrected visual acuities of 20/20. Optic neuritis was diagnosed based on the presence of acute visual symptoms, such as acute vision loss or visual field defects indicative of optic neuropathy with a relative afferent pupillary defect in the affected eye . A SnellLCN2 levels were measured in the plasma samples of the patients with optic neuritis and controls. Blood samples were collected on the day of optic neuritis diagnosis. High-dose intravenous steroid therapy was started after blood sample collection. Plasma LCN2 levels were measured using a commercially available Sandwich ELISA DuoSet kit  according to the manufacturer’s instructions. The assays were performed in 96-well plates using 100 μL of plasma (1:400 dilution) per the manufacturer’s instructions. For standardization, human recombinant LCN2 protein was used at concentrations ranging from 39.06 to 2500 pg/mL. The results were normalized to the total protein content of the plasma samples. All measurements were determined from duplicated assays and averaged the results of the two assays.Full-length human MOG (FL-MOG) cDNA was cloned into the KpnI and XbalI sites of the HG10364-UT pCMV3-MOG vector . All sequences were confirmed using a BigDye™ Terminator v3.1 cycle sequencing kit  and an Applied Biosystems 3730 DNA analyzer . The cloned FL-MOG plasmid or the empty vector plasmid was transfected into human embryonic kidney 293 (HEK293) cells  using Effectene transfection reagent . After 48 h, the cells were transferred into medium containing 100 μg/mL hygromycin . The hygromycin-resistant cells were isolated after 2 weeks and further cloned by limiting dilution. FL-MOG expression was confirmed through real-time polymerase chain reaction with the primer sets T7: 5′-TAATACGACTCACTATAGGG-3′ and BGH: 5′-TAGAAGGCACAGTCGAGG-3′.6 cells/mL. The cells were centrifuged at 4 °C for 1 h. All further steps were carried out at 4 °C. Patient serum (diluted 1:50 in FACS buffer) was mixed with 1.0 × 105 cells (100 µL). After rocking for 1 h, the cells were washed, and bound IgG was detected using Alexa Fluor 647 mouse anti-human IgG1 Fc antibody  for 30 min and analyzed using a BD FACS Aria III Flow cytometer . An IgG binding index, calculated as the ratio of the median AF647 fluorescence of green fluorescent protein–positive cells to that of green fluorescent protein–negative cells, of ≥2.5 was considered positive , 0.443–0.880, In this study, we investigated the correlation between plasma LCN2 levels and MOG-IgG titers in patients with optic neuritis. Plasma LCN2 levels were significantly higher in patients with MOG-IgG–positive optic neuritis than in controls and patients with MOG-IgG–negative optic neuritis. Additionally, plasma LCN2 levels were positively correlated with MOG-IgG titers in patients with optic neuritis. The LCN2 and MOG-IgG correlation supports the immune and inflammatory nature of MOG-IgG positive optic neuritis, as LCN2 has been strongly associated with neuroinflammation. Therefore, our results highlight the diagnostic value of plasma LCN2 levels for differentiating MOG-IgG–positive optic neuritis from MOG-IgG–negative optic neuritis.Our results are consistent with those from our previous study, in which we found that LCN2 expression in the optic nerve was highly upregulated after EAON induction with MOG immunization and that LCN2 deficiency attenuated the pathogenesis of EAON . LCN2 isMoreover, patients with MOG-IgG–positive optic neuritis experience more frequent recurrences with shorter durations between attacks than MOG-IgG–negative optic neuritis ,4,11. ThThe limitations of this study include its retrospective nature, relatively small sample size, and short follow-up duration. Moreover, all the participants were ethnically Korean; our results may not be applicable to other populations. Finally, because the cohort was drawn from a single tertiary center, referral bias toward patients with severe and recurrent disease is anticipated. Nevertheless, limited data are available regarding the association of plasma LCN2 levels with optic neuritis. We plan to investigate the molecular mechanism underlying the increased plasma LCN2 levels in patients with MOG-IgG–positive optic neuritis with a focus on clinical factors that may influence plasma LCN2 levels in patients with MOG-IgG associated disorders.We believe this is the first study that evaluated the plasma LCN2 levels and their correlation with MOG-IgG titers in patients with optic neuritis. We found that patients with MOG-IgG–positive optic neuritis had significantly higher plasma LCN2 levels than healthy controls and patients with MOG-IgG–negative optic neuritis. Additionally, plasma LCN2 levels were positively correlated with MOG-IgG titers in patients with optic neuritis. The LCN2 and MOG-IgG correlation supports the immune and inflammatory nature of MOG-IgG positive optic neuritis, as LCN2 has been strongly associated with neuroinflammation. Our results imply that measurement of plasma LCN2 levels may be helpful in differentiating MOG-IgG–positive optic neuritis from MOG-IgG–negative optic neuritis. In addition, measuring both plasma LCN2 levels and MOG-IgG titers could be useful for avoiding false positive results and overcoming the limitations of single-cell assays.


Plant cell walls are essential structures for plant growth and development as well as plant adaptation to environmental stresses. Thus, plants have evolved signaling mechanisms to monitor the changes in the cell wall structure, triggering compensatory changes to sustain cell wall integrity (CWI). CWI signaling can be initiated in response to environmental and developmental signals. However, while environmental stress-associated CWI signaling has been extensively studied and reviewed, less attention has been paid to CWI signaling in relation to plant growth and development under normal conditions. Fleshy fruit development and ripening is a unique process in which dramatic alternations occur in cell wall architecture. Emerging evidence suggests that CWI signaling plays a pivotal role in fruit ripening. In this review, we summarize and discuss the CWI signaling in relation to fruit ripening, which will include cell wall fragment signaling, calcium signaling, and NO signaling, as well as Receptor-Like Protein Kinase (RLKs) signaling with an emphasis on the signaling of FERONIA and THESEUS, two members of RLKs that may act as potential CWI sensors in the modulation of hormonal signal origination and transduction in fruit development and ripening. As each plant cell is tightly connected to its neighbors through shared walls, plant tissues form a continuum of cells . Thus, pArabidopsis, RLKs constitute a superfamily that contains more than 600 members, which can be again categorized into 12 subfamilies [Receptor-like kinases (RLKs) are serine/threonine kinases with extracellular domains to perceive the cell wall-associated signals ,8. Accorfamilies ,14,15. Nfamilies ,21,22,23families ,25,26,272+) signal, which has been well established to play critical roles in a diversity of biological processes. Plant cell walls have been regarded as extracellular Ca2+ pools, where Ca2+ is largely bound to pectin [2+ would substantially change along with fruit growth and development. Consequently, Ca2+ signaling should play an important role in fruit ripening. However, studies and reviews on the wall modification associated-Ca2+ signaling have been lacking.Besides RLKs, there exist two other groups of signals that may be tightly coupled with CWI signaling. One group derives from cell wall metabolites. For example, cellulose and the pectin metabolism-associated oligo-saccharide fragments have been demonstrated to be capable of triggering cellular signaling in response to CWI impairment caused by environmental stresses ,32,33,34o pectin ,36,37. AHormones, such as ethylene and abscisic acid (ABA), are the main determinants of fruit ripening. According to the physiological features of fruit ripening, fleshy fruits can be categorized into two major groups, i.e., climacteric fruits (CL) that show dramatic increases in respiration and ethylene production during fruit ripening, and non-climacteric fruits (NC) that do not show a typical increase in respiration and ethylene production ,39. It hFruit softening is the key event in fruit ripening. Fruit ripening is a highly coordinated and genetically programmed process involving a series of structural, physiological, and biochemical changes, finally leading to the development of edible ripe fruit with desirable quality attributes . SofteniFruit softening has been thought to result from cell wall modification. Besides, it has also been suggested to be affected by cell turgor ,49. In tVitis vinifera) was accompanied by an apoplastic solute accumulation, thus resulting in a dramatic decrease in cell turgor. ‘Delayed Fruit Deterioration’(DFD) is a tomato cultivar exhibiting dramatically delayed softening [The plant cell wall is a complex composite of polysaccharides and proteins, which can be again categorized into several broad classes, including cellulose, hemicellulose, pectins, expansins, etc. ,58,59. Toftening  and, theoftening .Solanum lycopersicum, named a colorless non-ripening mutation (Cnr) [Cnr has a phenotype characterized by the fruit that shows inhibition of softening, yellow skin, and a non-pigmented pericarp [Cnr phenotype is tightly coupled with a feature of much reduced cell-to-cell adhesion, principally owing to an enhanced cell separation in the fruit pericarp [Fragaria vesca), we observed that cell separation occurred from the very earliest stage of the fruit set. Moreover, the time course of the accelerated cell separation just coincided with the onset of ripening, implying an important role of cell separation in the initiation of strawberry fruit ripening [As it has long been known that fruit ripening is essentially regulated by hormonal signals, the change in cell metabolism during fruit ripening has been generally thought to be a result of hormonal signaling. In other words, the change in cell wall metabolism has been merely regarded as an event initiated by fruit ripening . Howeveron (Cnr) ,64. Cnr pericarp . This stWhile decades of research have attempted to prevent fruit softening through a delay of the entire ripening process ,50,58,66The fruit cell wall is principally composed of cellulose, hemicelluloses, pectin, and a small fraction of proteins . It has FaPG1 or FaPG2, induced the expression of a putative high-affinity potassium transporter. As the potassium transporter contributes to the promotion of cell turgor, it was proposed that the PGs’ modification of cell wall might be linked to the loss of cell turgor, which was again proposed to be the basis for the regulation of fruit firmness [Cell wall modification in ripening fruit is highly complex, involving diverse families of wall-modifying proteins . Among tfirmness . LogicalTo date, there are many attempts to modify softening through overexpression or silencing of genes encoding cell wall modifying proteins ,71,72, iPisum sativum) stem. Similarly, OGs were shown to inhibit auxin-induced root formation in tobacco and Arabidopsis leaf explants [FaPE1, a gene encoding pectin methyl esterase from F. ananassa, decreased the size of OGs extracted from the ripe F. vesca fruits, and application of the OGs from the transgenic fruits more strongly induced the expression of PR5, a maker gene of pathogen resistance, compared to that from the wild type. Moreover, ectopic expression of FaPE1 promoted the content of aromatic amino acids phenylalanine, tryptophan, tyrosine, and aspartate. More recently, Yang et al. [Botrytis cinerea inhibited fruit softening in ripening tomatoes. This research suggest that OGs are indeed capable of serving as signals to regulate plant growth and development. However, to date, studies on changes in endogenous OGs in fruit growth and ripening have been lacking. Accordingly, it can’t currently be concluded that OGs are key signals implicated in fruit ripening.In the past decades, studies on CWI signaling have been mainly focusing on plant immunity. Pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) have been identified to be primary signals to trigger plant immunity responses ,77,78,79explants ,83. In sexplants  reportedg et al.  reported2+ is well known as a second messenger that plays powerful roles in diverse biological processes. One of the bases for Ca2+ to act as a signal is its compartmentalized distribution with cell walls as a pool where the content of Ca2+ may be ten thousand times higher than the cytoplasm, such that, in responses to stimuli, Ca2+ can quickly enter into cells to initiate downstream signaling [2+ is largely bound to pectin [2+, although there is a report that the total content of the extracellular Ca2+ was not significantly altered during fruit ripening [2+ on fruit surface is capable of significantly affecting fruit ripening [2+ in the apoplast, as a result of pectin degradation, would expect to have an impact on fruit ripening.Caignaling ,36,37. Io pectin ,87. Consripening . Pharmacripening ,89,90, i2+ signaling-associated components, such as the Ca2+ sensor, calmodulin, Ca2+-dependent protein kinases, and CDPK, might be implicated in fruit ripening [2+/calmodulin-regulated SR/CAMTAs was dramatically altered in ripening mutant rin compared with wildtype fruit, and moreover, transiently overexpressing SlCaM2 in mature green fruit delayed ripening, while reducing SlCaM2 expression accelerated ripening, implying involvement of Ca2+/Calmodulin-associated signaling in the regulation of fruit development and ripening [2+ signaling acts to interact with hormonal signaling, jointly regulating fruit ripening [2+ signaling plays an important role in the regulation of fruit ripening. But from the point of view of CWI signaling, it remains unclear whether the cell wall modification may alter the content of extracellular free Ca2+ and whether this alteration may contribute to the regulation of fruit ripening. To conclusively demonstrate that Ca2+ signaling is involved in fruit ripening, investigations on the change of extracellular free Ca2+ along with fruit development and ripening and its effect on fruit ripening are required.In support of pharmacological studies, molecular research has increasingly suggested that many Caripening ,93,94,95ripening ,96,97. TNitric oxide (NO) has been well established to play a crucial role in plant growth and environmental adaptation ,99. As aCarica papaya L. cv. ‘Sui you 2′) [Brassica oleracea), green bean (Phaseolus vulgaris) [Capsicum annuum L.) [A pioneering study by Leshem et al.  reported you 2′)  or even ulgaris) , pepper nuum L.) . In suppnuum L.)  demonstrnuum L.) ,117. Impnuum L.) ,119,120.Arabidopsis, Rasul et al. [PER4) and a β-1,3-glucanase. Importantly, an S-Nitroso-Proteome study by Navarro et al. (2020) demonstrated that, in response to nitrosative stress, a large number of proteins were S-nitrosylated, among which several proteins are involved in CWI signaling, such as Hsp60 and Hsp90 as well as the Arp2/3 complex subunit that has been suggested to regulate chitin and β-glucans metabolism thereby affecting CWI in Candida albicans [fer-4 mutant lacked comparable activity of NO accumulation. These observations provide direct support for a signaling linkage between FER and NO.As mentioned above, OGs play important roles in CWI signaling. There is evidence that OGs were capable of triggering NO production in many biological processes. For example, a study by Hu et al.  (2003) rl et al.  (2012) dalbicans ,125. Ovealbicans  (2022) dArabidopsis [Arabidopsis, the CrRLK1L subfamily contains 17 members, many of which have been characterized, e.g., THESEUS1 (THE1), HERCULES1 (HERK1), HERCULES2 (HERK2), FERONIA/SIRENE (FER/SIR), ANXUR1 (ANX1), ANXUR2 (ANX2), etc. and among these characterized members, FER and THE have attracted particular interest, owing to their special roles in CWI signaling [Plant receptor-like protein kinases (RLKs) belong to an evolutionarily conserved superfamily with more than 600 members in bidopsis ,128. Thebidopsis . The RLKbidopsis . Among tbidopsis . Membersignaling ,132,133.ignaling ,14,15,23Arabidopsis mutant feronia (fer), the pollen tube fails to arrest and thus continues to grow inside the female gametophyte without rupture for the release of sperms [FER was named for the Etruscan goddess of fertility due to its important role in reproduction . The mecf sperms ,18. Besif sperms ,136,137,f sperms ,141,142,f sperms ,145,146,MdFERL6 in apple fruit callus inhibited and promoted ethylene production, respectively. By using virus-induced gene silencing (VIGS), it was found that down-regulation of SlFERL1, a homolog gene of MdFERL6, promoted tomato fruit ripening. Moreover, MdFERL6 and MdFERL1 could physically interact with MdSAMS (S-adenosylmethionine synthase), a key enzyme in the ethylene biosynthesis pathway, implying that FER may regulate apple and tomato fruit ripening vial modulation of ethylene production. In strawberries (Fragaria × ananassa), we found that overexpression and RNAi-mediated downregulation of FaMRLK47, a member of FER, delayed and accelerated fruit ripening, respectively. FaMRLK47 could physically interact with FaABI1, a negative regulator of abscisic acid (ABA) signaling, implying that FER plays an important role in the regulation of strawberry fruit ripening [SlFERL (FERNOIA-like) to activate SlFERL expression. Importantly, overexpression of SlFERL significantly accelerated the ripening process of tomato fruit, whereas RNA interference knockdown of SlFERL delayed fruit ripening. Although, to date, there is scarce research on FER signaling in relation to fruit ripening, the existing evidence conclusively demonstrates that FER should be a key signal controlling both CL and NC fruit ripening.FER contains malectin-like domains that have been demonstrated to be capable of interacting with cell-wall polysaccharides, such as pectin, which led to the speculation that it can potentially function in CWI signaling. Because cell wall degradation is a key event accompanied by fruit ripening, it is reasonable to propose that FER may likely play a critical role in fruit ripening. In accordance with this proposal, we previously demonstrated that FER plays an important role in fruit ripening ,25,26. Iripening . More reripening  reported2+, NO, and ROS are potential signals implicated in the regulation of fruit ripening. Strikingly, it has been increasingly suggested that FER signaling may act to trigger the release of these signals. For example, a study by Gjetting SK et al. [Arabidopsis, and more recently, a study by Gao et al. [2+ signaling for pollen tube reception. Also, a study by Luo et al. [As discussed above, CaK et al.  (2020) ro et al.  (2022) do et al.  (2022) ro et al.  (2020) do et al. ,150,151.ABA signalingArabidopsis, FER signaling is initiated by an interaction of the FER kinase with guanine exchange factors, such as GEF1, GEF4, and GEF10, which subsequently activates GTPase ROP11/ARAC10. A study by Yu et al. [Arabidopsis mutants disrupted in any step of the FER signaling pathway displayed an ABA hypersensitive response. Further studies demonstrate that the ROP11/ARAC10 protein physically interacted with the ABI2 phosphatase and enhanced its activity. Intriguingly, besides ROP11/ARAC10, a subsequent study by Chen et al. [Hormones are well known to be the major determinant of fruit ripening, among which ethylene acts to control CL fruit ripening, whereas a synergistic action of ABA, IAA, JA, and BR act to control NC fruit ripening ,43,44,45u et al.  reportedn et al.  demonstrIAA signalingfer-4 mutant seedlings displayed a substantial resistance to apoplast alkalization. fer-4 mutant displays increased lateral root branching and delayed gravitropic response, which is associated with polar auxin transport (PAT). Moreover, the fer-4 mutant resulted in asymmetric root growth, which can be prevented by suppressing the polar auxin transport in fer-4 [IAA is a major regulator of cell growth . Studiesin fer-4 ,165. TheBR signalingFER as well as two other tightly linked genes, HERCULES1 and THESEUS1, are target genes of BR signaling, as evidenced by those observations that BZR1 could bound to the promoters of FERONIA2 (FER2) and FER3 whereby directly inducing their expression, and moreover, fer knockout mutant shows altered responsiveness to BR [BR receptor, BRI1, belongs to receptor-like kinase BRASSINOSTEROID INSENSITIVE 1. Upon BR ligand binding, BRI1 and its co-receptor BAK1/somatic embryogenesis receptor kinase (SERK)3 transphosphorylation each other, initiating a signaling cascade leading to the regulation of transcription factors BZR1 and BES1/BZR2 . A numbess to BR . OverallJA signalinglrx345 triple mutant displays many similar phenotypes with the fer-4 mutant, suggesting that FER signaling may be linked to the functional regulation of extensins. In the lrx345 triple mutant, many JA and SA-responsive genes are constitutively up-regulated, whereas disruption of the JA pathway, via crossing lrx345 to the coi1-1 mutant, suppressed the dwarf phenotype of the lrx345 and fer-4 mutants, implying the functional of extensin by FER is correlated to JA signaling. More recently, Darwish et al. [fer-4 mutant.JA signaling pathway consists of COI1, JAZ, and MYC2. COI1 was identified as the JA receptor, which is essentially a ubiquitin ligase, targeting the JASMONATE ZIM-domain (JAZ) repressor for degradation by the 26S proteasome . As a trh et al.  reportedEthylene signalingArabidopsis mutants with increased ethylene response revealed a transferred DNA (T-DNA) insertion in the coding sequence of FER, suggesting an important role of FER in ethylene signaling [fer mutants led to a dwarf phenotype with an elevated level of SAM and ethylene. Jia et al. [MdFERL6 or MdFERL1, the apple homolog of Arabidopsis FER, fruit delayed ripening, and suppressed ethylene production. Virus-induced gene silencing (VIGS) of SlFERL1, the tomato homolog of FER, promoted tomato fruit ripening and ethylene production. Both MdFERL6 and MdFERL1 were found to be capable of physically interacting with S-adenosylmethionine synthase, further demonstrating the role of FER in the regulation of fruit ripening via modulation of ethylene production [SlFERL and activate its expression. Thus, FER and Ethylene signaling may constitute a loop of modification, i.e., while FER acts to modulate ethylene biosynthesis, ethylene signaling functions to regulate FER expression, thereby promoting FER signaling.A decade ago, a screen for ignaling . A subseignaling  demonstra et al.  reportedoduction . RIPENINoduction  reportedArabidopsis in the mutation background of cellulose synthase CESA6 [the1-1  and enhanced in the1-4  seedlings upon treatment with active Driselase compared to Col-0 seedlings, suggesting that THE1 was implicated in the CWI impairment-induced JA accumulation [herk1 and the1-4 strongly responded to salt stresses, implying that THE1 plays a role in stress response via regulating PME metabolism. These observations demonstrate that THE1 can be a key sensor of CWI.THE1 was discovered in a screen for suppressors that attenuated the short hypocotyl phenotype of dark-grown se CESA6 . As a rese CESA6 ,177,178.se CESA6  reportedmulation . Very remulation  reportedherk1 the1 double mutant partially suppressed the cell elongation phenotype of bes1-D, a gain of function mutant that displays constitutive BR responses, suggesting that HERK1 and THE1 cooperate with BR pathway and mediate part of BR-regulated cell elongation. In addition, Nikonorova et al. [ralf34-1 mutant, a ligand of THE1, displayed reduced sensitivity to NAA in its growth-suppressing responses, implying that THE1 is implicated in the modulation of IAA signaling. More importantly, a recent study by Bacete et al. [the1-1  and the1-4  mutant seedlings compared to Col-0 controls. Meanwhile, it was found that the sorbitol-induced ABA accumulation was enhanced in the1-4 seedlings compared to Col-0 controls, suggesting that THE1 acted as a negative regulator of ABA biosynthesis. The mechanism for dehydration-induced ABA accumulation has long been a key and intriguing research topic for plant scientists, but so far, little is known about it. The findings that THE acts to mediate ABA biosynthesis is a breakthrough in the research on the ABA signal production in response to developmental and environmental stimuli. As both ABA and JA are key regulators of fruit ripening, it is of great significance to explore whether THE signaling is implicated in fruit ripening.Although much less is known about the role of THE in hormonal signaling compared to that of FER, a number of studies suggest that THE1 indeed plays pivotal roles in both hormonal biosynthesis and signal transduction. For example, a study by Guo et al.  showed ta et al.  reportede et al.  reported2+ signaling, and OGs signaling potentially play pivotal roles in the initiation of fruit ripening. NO potentially acts as a signal downstream of OGs and FER and has been established to modulate ethylene biosynthesis. Hormones, especially ethylene and ABA, are well-known internal cues controlling fruit ripening. Acting as important sensors of CWI, FER, and THE have been increasingly demonstrated to be capable of modulating hormone biosynthesis and signal transduction. To further unravel the molecular mechanism for the control of fruit ripening, it is of particular significance and importance to explore the following aspects:2+ signaling has been well demonstrated to play important roles in fruit ripening, but it is largely unknown how Ca2+ signaling is initiated. As cell walls serve as an extracellular pool of Ca2+, degradation of the walls would expect to cause a change in the level of free Ca2+, thereby producing an impact on Ca2+ signaling. Investigation of the changing pattern of free Ca2+ as affected by cell wall degradation would contribute to a profound understanding of the signaling mechanisms for the regulation of fruit ripening.CaArabidopsis, FER and THE have been suggested to play an important role in the biosynthesis of multiple hormones, such as ethylene, ABA, and JA. Further studies are required to demonstrate whether FER and THE may function in hormonal production during fruit ripening.Hormones are well known to be key regulators of fruit ripening. However, little is known about the mechanisms for their origination, along with fruit development and ripening. In As CWI sensors, FER and THE have been increasingly suggested to play important roles in modulating multiple hormone signaling via cross-talk between CWI and hormonal signaling. Conversely, many hormones have been suggested to be capable of modulating FER and THE-associated signaling via regulating the expressions of FER and THE. Thus there exists a reciprocal modulation between hormone and CWI signaling, reflecting the complexity of the mechanisms for the regulation of fruit ripening. Investigation of the mechanisms for the reciprocal modulation between hormone and CWI signaling would contribute to a better deciphering of the signaling networking controlling fruit ripening.


Chronic kidney disease (CKD) is a major global health problem for which there are no curative drug treatments. Hyperuricemia is one of risk factors for CKD. The evidence on effects of uric acid (UA)-lowering treatments on the progression of CKD was very limited and previous meta-analyses used only trials which primarily used xanthin oxidase (XO) inhibitors because the reports on fulminant hepatitis due to benzbromarone kept us from using uricosuric agents for hyperuricemia patients. Dotinurad, a novel selective urate reabsorption inhibitor for the treatment of hyperuricemia, reduces serum UA levels by selectively inhibiting urate transporter 1 (URAT1). We retrospectively picked up patients who had taken dotinurad from June 2018 to August 2021 and compared metabolic parameters at baseline with the data at 3 and 6 months after the start of dotinurad. We found 84 patients, and approximately 74% of patients were complicated with CKD. After the start of dotinurad, improvements in serum lipids, systolic blood pressure, body weight, and albuminuria, in addition to reduction in serum UA, were observed. Dotinurad increased urinary UA excretion, and was effective to reduce serum UA in patients with both UA underexcretion type and renal UA overload type. Furthermore, urinary UA excretion was significantly and negatively correlated with serum creatine levels at baseline and at 6 months after the start of dotinurad, and the change in urinary UA excretion after 3 months was significantly and negatively correlated with change in serum creatine levels. The property of dotinurad, which selectively inhibits URAT1, but not other UA transporters, such as ATP-binding cassette, subfamily G, and 2 (ABCG2), which ABCG2 is a UA and uremic toxin exporter, may be beneficially associated with pathology of CKD. URAT1 can be a therapeutic target molecule for CKD and DKD. Chronic kidney disease (CKD) is a major global health problem for which there are no curative drug treatments. Lifestyle-related diseases, such as obesity, hypertension, diabetes, and dyslipidemia, are currently important risk factors for CKD . EvidencHyperuricemia is also a lifestyle-related disease, but most patients with hyperuricemia have no symptoms. Some studies recommend lower serum uric acid (UA) values < 5 mg/dL for the apparently healthy population, while the European League Against Rheumatism recommends a target of <6.0 mg/dL with medication for anti-hyperuricemia ,13,14. CA case–control study of 100 patients with primary gout and 72 healthy controls was undertaken . Most pan = 80) with or without gout was performed [Dotinurad, a novel selective urate reabsorption inhibitor (SURI) that has a potential for the treatment of hyperuricemia, reduces serum UA levels by selectively inhibiting urate transporter 1 (URAT1) . The RCTerformed , and doterformed .Here, we aimed to study the Influences of dotinurad on metabolic parameters, including the markers for renal function and UA excretion in patients with hyperuricemia.The study protocol was approved by the Ethics Committee of the National Center for Global Health and Medicine (NCGM-S-004615-00), and the study was performed in accordance with the Declaration of Helsinki.2) or proteinuria including albuminuria .We retrospectively picked patients who had taken dotinurad for the treatment of asymptomatic hyperuricemia, from September 2021 to December 2022, and compared metabolic parameters at baseline with the data at 3 and 6 months after the start of dotinurad. Information about medical history and medication were obtained via an electronic medical record. Such information included age, gender, body weight, body mass index (BMI), systolic and diastolic blood pressures, comorbidities, and treatments for type 2 diabetes, hypertension, dyslipidemia, and hyperuricemia. Informed consent was obtained by the opt-out approach. According to the diagnostic criteria by the Japan Diabetes Society, the Japanese Society of Hypertension, Japan Atherosclerosis Society; Japanese Society of Gout and Uric and Nucleic Acids, we defined type 2 diabetes as taking anti-diabetic drugs and/or HbA1c > 6.5%; hypertension as taking anti-hypertensive drugs and/or systolic blood pressure ≥140 mmHg and/or diastolic blood pressure ≥90 mmHg; dyslipidemia as taking anti-hyperlipidemic drugs and/or low-density lipoprotein-cholesterol (LDL-C) ≥ 140 mg/dL and/or triglyceride (TG) ≥ 150 mg/dL and/or triglyceride high-density lipoprotein-cholesterol (HDL-C) < 40 mg/dL; hyperuricemia as taking uric acid (UA) lowering drugs and/or serum UA ≥ 7.0 mg/dL, respectively. According to the diagnostic criteria by the Japanese Society of Nephrology, CKD was defined as reduced eGFR , aspartate aminotransferase (AST), and gamma-glutamyl transferase (GGT) were measured by a modified Japan Society of Clinical Chemistry (JSCC) reference method. Serum albumin was measured by the modified bromocresol purple (BCP) method. Serum UA was measured by uricase peroxidase method. Plasma glucose was the hexokinase UV method. Hemoglobin A1c (HbA1c) was measured by automated enzyme-linked immunosorbent assays . Serum creatinine, TG, HDL-C, and LDL-C were determined enzymatically. The eGFR was calculated by using the Chronic Kidney Disease Epidemiology Collaboration (CKD–EPI) formula. To understand urinary excretion of UA, we measured urinary UA/creatinine.Hyperuricemia has been previously classified into the “UA overproduction type”, “UA underexcretion type”, and “combined type” . ATP-binAnti-hypertensive drugs, such as angiotensin receptor blockers (ARB), and anti-hyperlipidemic drugs, such as statin and SGLT2 inhibitors , may affp value of <0.05 and <0.1 was considered to be statistically significant, and to have tendency, respectively.Statistical analyses were performed by using SPSS version 23 . All values are expressed as the mean  ±  standard deviation, except for sex. The paired t-test was used to statistically analyze comparison in metabolic parameters between before and after the start of dotinurad. Correlations between changes in two parameters were statistically analyzed by the Spearman’s correlation (non-parametric). 2, indicating that our study included a relatively large number of overweight patients. Almost 60% of patients had type 2 diabetes, hypertension, and dyslipidemia. Very high-percentage (approximately 74%) of patients were complicated with CKD.We found 84 patients, and baseline characteristics for patients who had taken dotinurad were shown in Thirty-five patients had not used UA-lowering drugs, and forty-nine patients had undergone the switching from XO inhibitors, such as febuxostat, topiroxostat, and allopurinol, to dotinurad. A half of patients had taken daily 0.5 mg of dotinurad, and 44% of patients had taken daily 1.0 mg of dotinurad.Treatments for hypertension, dyslipidemia, and type 2 diabetes at baseline were shown in Changes in metabolic parameters after the start of dotinurad were shown in Changes in metabolic parameters after the start of dotinurad in patients who had not used UA-lowering drugs were shown in Body weight, blood pressures, transaminases, and glucose metabolism did not show any changes. Serum LDL-C significantly decreased and non-HDL-C tended to decrease at 3 months after the start of dotinurad as compared with baseline. Serum UA significantly decreased at both 3 and 6 months after the start of dotinurad. UACR tended to decrease at 6 months after the start of dotinurad.Changes in metabolic parameters after the start of dotinurad in in patients who switched from XO inhibitors were shown in Body weight tended to decrease at 6 months after the start of dotinurad as compared with baseline. Systolic blood pressure significantly decreased at 3 months after the start of dotinurad as compared with baseline. Transaminases, glucose, and lipid metabolism, eGFR and UACR did not show any changes. Serum UA significantly increased at 3 months after the start of dotinurad as compared with baseline; however, serum UA did not show a significant difference at 6 months after the start of dotinurad.Changes in urinary UA/creatine after the start of dotinurad in patients were shown in Changes in metabolic parameters after the start of dotinurad in patients who had taken anti-hypertensive drugs  were shown in Changes in metabolic parameters after the start of dotinurad in patients who had not taken anti-hypertensive drugs were shown in Changes in metabolic parameters after the start of dotinurad in patients who had taken anti-hyperlipidemic drugs  were shown in Changes in metabolic parameters after the start of dotinurad in patients who had not taken anti-hyperlipidemic drugs were shown in Changes in metabolic parameters after the start of dotinurad in patients who had also taken SGLT2 inhibitors were shown in Changes in metabolic parameters after the start of dotinurad in patients who had not taken SGLT2 inhibitors were shown in Changes in metabolic parameters after the start of dotinurad in patients who had not taken anti-hypertensive and anti-hyperlipidemic drugs and SGLT2 inhibitors were shown in The proportion of hyperuricemia phenotype in our patients were shown in The effect of dotinurad on urinary UA/creatinine and serum UA levels in each hyperuricemia phenotype were shown in Correlations between urinary UA/creatinine and serum creatinine were shown in Correlations between urinary UA/creatinine and eGFR were shown in Correlations between serum UA and serum creatinine were shown in Correlations between serum UA and eGFR were shown in Almost 60% of our patients had type 2 diabetes, and over 70% of patients have CKD; therefore, present study could observe the effect of a novel selective URAT1 inhibitor, dotinurad on metabolic and renal parameters in CKD and DKD patients. In other words, this study may clarify a clinical significance of URAT1 in CKD and DKD. Renal excretion of UA is the major regulator of serum UA concentration ,29,30. IWe found that dotinurad improved UACR in addition to serum UA in all patients and patients who had not used UA-lowering drugs. An increase in urinary UA excretion measured by urinary UA/creatine can indicate the inhibitory effect of URAT1 by dotinurad. Dotinurad significantly increased urinary UA/creatine in all patients, especially in patients who switched from XO inhibitors. As almost 88% of our patients were UA underexcretion type, such patients should have been treated by uricosuric agents. As the effect of dotinurad on urinary UA/creatinine in each hyperuricemia phenotype showed, dotinurad significantly increased urinary UA/creatinine in patients with UA underexcretion type. More interesting is the effect of dotinurad on urinary UA/creatinine and serum UA levels in patients with renal UA overload type. In patients with renal UA overload type, there were no significant changes in urinary UA/creatinine by dotinurad. However, dotinurad remarkably reduced serum UA as compared with baseline. On the other hand, in patients with UA underexcretion type, despite significant increase in urinary UA/creatine, the decrease in serum UA levels was modest. This suggests that expression and/or function of URAT1 in patients with UA underexcretion type may be higher than those in patients with renal UA overload type. In short, while the same dose of dotinurad increases urinary UA excretion, the effect on serum UA levels may be insufficient in patients with UA underexcretion type with higher expressed URAT1; the same dose of dotinurad increases urinary UA excretion, but the effect on serum UA levels may be greater in patients with renal UA overload type with lower expressed URAT1. The increased protein level of URAT1 was observed in obesity/metabolic syndrome model mice . Upon hiWe analyzed the correlation of urinary UA/creatinine with the markers for CKD/DKD, such as serum creatinine levels and eGFR. Urinary UA/creatinine was strongly associated with lower serum creatinine levels and higher eGFR, and the increase in urinary UA/creatinine by dotinurad was also associated with an improvement of serum creatinine levels and eGFR. However, the influence of serum UA on serum creatinine levels was weak, and serum UA levels were not associated with eGFR. This suggests that urinary UA excretion may perform a more crucial role in progression of CKD/DKA as compared with serum UA levels.We found an improvement in serum lipids, such as reduction in LDL-C and non-HDL-C by dotinurad, in patients who had not used UA-lowering drugs. The meta-analysis showed that hyperuricemia increased the likelihood of dyslipidemia, and the pooled OR for the highest UA level vs. the lowest UA level was 1.84  . A reducWe adjusted confounding variables, such as concurrent medications, for other lifestyle-related diseases except for hyperuricemia because such treatments have an effect on CKD outcome and metabolic parameters. Hyperuricemia was strongly associated with metabolic syndrome  and metaAnti-hypertensive drugs, such as ARB and calcium channel blockers, have a favorable impact on blood pressure and renal parameters for CKD ,48 FiguA. TherefAnti-hyperlipidemic drugs, such as statins, fibrates, and ezetimibe, have beneficial effects on dyslipidemia and CKD ,50,51,52SGLT2 inhibitors have beneficial effects on hyperuricemia, dyslipidemia, glucose metabolism, blood pressure, and CKD  Figure A. DiastoBody weight, HbA1c tended to decrease and eGFR tended to increase in patients who had not taken anti-hypertensive and anti-hyperlipidemic drugs, and SGLT2 inhibitors, supporting a significance of a relative inhibition of GLUT-9 by dotinurad for an improvement of metabolic and renal parameters.The summery of effects of dotinurad with and without concurrent medications for other lifestyle-related diseases except for hyperuricemia were shown in ABCG2 is a high-capacity UA exporter, the dysfunction of which raises hyperuricemia risk  Figure . DotinurLimitations of the study need to be addressed. This is a cross-sectional study, limiting inferences of causality and its direction. Although we did not change treatments for diabetes and hypertension, dyslipidemia intentionally during the study period, we cannot deny the beneficial role of the concomitant assumption of other drugs including aspect of synergism and/or the possible interaction between dotinurad and other treatments for metabolic parameters. We classified hyperuricemic patients into “UA overproduction type” and “UA underexcretion type” based on urinary UA/creatinine ≥ or <0.5, which may be weak. We should have measured 24 h urine UA, and should have analyzed urate transporter variants. To elucidate this in further studies, preferentially, RCT, that including a large number of patients, should be performed in the future.Dotinurad, a selective URAT1 inhibitor, improved serum lipids, blood pressure, body weight, and albuminuria, in addition to reduction in serum UA, in CKD/DKA patients. Present study suggested that dotinurad was effective to reduce serum UA by using appropriate dose in patients with both UA underexcretion type and renal UA overload type. Furthermore, present study suggested that an increase in urinary UA excretion was favorably associated with renal function. The property of dotinurad, which selectively inhibits URAT1, but not ABCG, may be beneficially associated with pathology of CKD. URAT1 can be a therapeutic target molecule for CKD and DKD.


Lactobacillus plantarum, and L. buchneri on chemical composition, fermentation, aerobic stability, dynamics of microbial community, and metabolic pathway of a mixture of lucerne, wheat bran (WB), and rice straw (RS). All mixtures were ensiled for 1, 3, 5, 7, 15, 30, and 45 days after treatment with uninoculated ; L. plantarum ; L. buchneri ; LP + LB ; and pre-fermented juice . Four lactic acid bacteria (LAB) species from three genera were cultured from the pre-fermented juice, with W. cibaria being dominant. The inoculants increased lactic acid (LA), decreased pH and ammonia nitrogen (AN) compared to C silage at earlier stages of ensiling, and high dry matter (DM) and water-soluble carbohydrate (WSC) content in inoculated silages. Adding LPB increased the abundance of L. plantarum, L. paralimentarius, and L. nodensis, resulting in the lowest pH. Pre-fermented juice enriched W. cibaria, L. sakei, L. parabrevis, Pseudomonas putida, and Stenotrophomonas maltophilia, mainly enhanced accumulation of acetic acid (AA) and LA, and decreased pH, crude protein losses, AN, and hemicellulose contents. L. buchneri and L. brevis had a high abundance in LB-treated and J silages, respectively, inhibited undesirable bacteria, and improved aerobic stability with more than 16 days. In addition, the metabolic pathways changed with time and L. buchneri inoculants promoted global metabolism. In conclusion, inoculations altered bacterial succession and metabolic pathways in silage; LB and pre-fermented juice enhanced ensiling by promoting pH reductions, enhancing concentrations of LA and AA, and extending aerobic stability more than 16 days.The objective of this study was to determine the effect of pre-fermented juice,  Lactobacillus plantarum (LP) to become the dominant microbe. Furthermore, WB can also be combined with RS to produce high-nutrient silage by increasing DM recovery  for silage. Fortunately, crop by-product, an important biomass resource in green, sustainable, and intensive agricultural production, is a promising adsorbent to reduce silage moisture. Ensiling lucerne with crop by-products would not only reduce moisture, but also reduce competition between humans and animals for the same food sources . Rice strecovery . Thereforecovery .L. plantarum and L. buchneri to decrease pH and inhibit yeasts and molds , and pre-fermented juice. The latter was prepared from lucerne that was chopped from three plots and used as replicates. For this, 200 g of chopped forage were mixed with 1 L of distilled water and macerated for 2 min in a blender, filtered (two layers of cheesecloth), and glucose (2 g/100 ml) was added, the solution was well mixed and placed in a sealed plastic container in an incubator at 30°C for 48 h , whereas WB was from Kangda Agricultural Products Co., Ltd. . In May 2019, the second crop of lucerne at the 10% bloom stage was harvested (hand clippers) from three plots. The RS was procured after rice was harvested. Lucerne and RS were processed in a fodder cutter and reduced to an overall length of 1–2 cm. Lucerne, WB, and RS were combined in a ratio of 80:15:5, based on preliminary experiments. Additives used were commercial for 48 h .L. plantarum ; (3) L. buchneri ; (4) LP + LB ; (5) pre-fermented juice . Inoculants were added to deionized water, and with constant mixing, 20 ml/kg was sprayed as a fine mist on the material to be ensiled. Similarly, 20 ml/kg of fermented juice or distilled water , were also applied. All samples  were ensiled in vacuum-heat sealed nylon-polyethylene standard barrier bags . Air was withdrawn from silos; they were sealed with a vacuum extractor  and kept at ∼30°C. On Days 1, 3, 5, 7, 15, 30, and 45, triplicate samples were collected to assess fermentation and composition. Assessment of the microbial community was done at Day 5, 15, and 45 of ensiling and for the fermented juice, after 48 h of incubation.A total of 158 silage mixtures  were prepared, with 105 randomly selected (5 treatments × 7 sampling days × 3 replicates/treatment), as follows: (1) uninoculated ; (2) via an Agilent 1260 HPLC system with a UV detector and Acclaim TM organic acid column  with 50 mmol/L NaH2PO4, 0.6 ml/min at 30°C; and ammonia nitrogen (AN) (A sample of silage (20 g) was put into 180 ml distilled water and processed in a blender for 1 min. The solution was filtered through 2 layers of cheesecloth and pH measured . The filtrate was centrifuged  and the supernatant was assessed for the following: volatile fatty acids (VFAs), with gas chromatography , as described by gen (AN) . Samplesgen (AN) , crude pgen (AN) , and watgen (AN) , with ADgen (AN) .1 was used for data analysis.Pre-fermented juice samples (50 ml) were centrifuged at 10,000 × g for 5 min, the supernatant removed, and the residue stored at −80°C. The DNA was isolated  according to the manufacturer’s instructions, from frozen-thawed samples of pre-fermented juice and silage. The DNA was quantified with a NanoDrop 2000 and quality determined with 1% agarose gels. Single-molecule real-time (SMRT) sequencing was done, with primers 27F and 1492R to detect 16S rRNA genes, and polymerase chain reaction (PCR) was done as described by To determine aerobic stability, 90 piles of silage were prepared as described for silage, and 60 were randomly chosen and ensiled. After 45 days, bales were opened, mixed thoroughly, and loosely packed into 2-L sterile plastic boxes that were covered with two layers of gauze and held at 30–35°C. Every 2 h, temperatures of air and silage (middle of the bottle) were measured . Aerobic stability was defined as the interval for silage to become at least 2°C warmer than air. Effects of aerobic exposure on pH were determined after Day 0, 4, 8, 12, and 16 (three boxes per treatment).P < 0.05 was considered significant and differences were located with Tukey’s.This experiment was conducted as a completely randomized design . The GLM procedure  was used to conduct two-way ANOVA, with fixed effects of treatment, ensiling day, and their interaction. Fixed effects of LP, LB, LPB, and pre-fermented juice were used to assess sequence, bacterial diversity, and succession of inoculations. For all analyses, P < 0.05) in RS than lucerne, whereas the CP content was lower (P < 0.05) in RS than lucerne. The WB had higher (P < 0.05) DM and HC contents, but lower (P < 0.05) NDF, ADF, and CP content than lucerne. However, the mixture had lower (P < 0.05) NDF, ADF, and CP and higher (P < 0.05) DM and HC compared to lucerne  on pH, LA, AA, propionic acid (PA), butyric acid (BA), and AN, and there was an interaction between group and days for silage pH, LA, AA, propionic acid (PA), butyric acid (BA), and AN (P < 0.05) after 3 days of ensiling and was lower than C silage at Day 45. After 15 days of ensiling, pH of LPB silage was always lower (P < 0.05) than in the other four groups.There were effects of treatment group and ensiling days  LA concentration among all silages during the initial 7 days of ensiling, whereas the lower LA (P < 0.05) concentration in J silage than in C occurred at Day 15 and 30 of ensiling. The LA concentration of LB silage increased most slowly during 3 days of ensiling, and was significantly lower than all other groups at 45 days of ensiling. However, inoculated treatments with pre-fermented juice, LP, LB, or LPB had greater AA concentrations than the uninoculated C group at 45 days of ensiling. Concentration of AA was highest (P < 0.05) in J silage at both 3 and 15 days of ensiling. Furthermore, in LB and LPB silages, AA had the slowest (P < 0.05) increase in the first 5 days of ensiling, but AA in these two groups generally exceeded that in C silage until the end of the trial. The PA concentration was higher (P < 0.05) in J silage than LB silage only was observed after Day 5 of ensiling, and then it became lower (P < 0.05) than LB silage. Both LB and LPB silages had lower (P < 0.05) BA concentrations than other treatments after 45 days of ensiling. All inoculants reduced (P < 0.05) AN concentration relative to C silage after 1 day of ensiling. The addition of pre-fermented juice lowered AN concentration as compared to C silage over the entire ensiling, with LB silage having the opposite trend.There was rapid LA production in silage treated with pre-fermented juice, LP, LB, or LPB after 1 day of ensiling. The J silage had the highest (P < 0.01) on DM, WSC, CP, NDF, and HC and an interaction for DM, WSC, CP, NDF, and HC. The C silage had lower DM after 45 days of ensiling and tended to have lower WSC contents than other treatments after 7 days of ensiling. The content of WSC in J silage was lowest in the first 5 days of ensiling, with J silage having higher CP and lower HC contents than other treatments after 45 days of ensiling. The contents of NDF and HC decreased markedly in all groups at 45 days of ensiling with no differences in NDF and ADF content in inoculant treatments vs. control after 45 days of ensiling (There were effects of group and time (ensiling .P < 0.05) those of silage samples after 45 days of ensiling  in 9 raw materials and 45 silages samples; based on 3% dissimilarity, there were 306 operational taxonomic units (OTUs). The Simpson, Shannon index, and Chao1 values in raw materials were greater than , Acinetobacter sp. (12.8 and 9.7%), Sphingobacterium sp. (5.2 and 4.8%), uncultured bacterium Stenotrophomonas (4.4 and 5%), Pseudomonas putida (2.8 and 4.7%), and Pseudomonas fragi (4.8 and 4.9%) were the major bacterial species in lucerne and mixture (Weissella (97%), Lactobacillus (1.1%), Pediococcus (0.4%), Pantoea (0.4%), Pseudomonas (0.1%), and Sphingomonas (0.1%). The major microbiota species in pre-fermented juice were W. cibaria (96.9%), L. brevis (0.6%), Pantoea agglomerans (0.4%), L. plantarum (0.3%), L. paralimentarius (0.3%), and Pseudomonas putida (0.1%). After 5 and 15 days of ensiling, LA-producing bacteria  were the dominant genera (64.1–84.3%) in all silage samples. Furthermore, for all groups, at 5 and 15 days of ensiling, dominant LAB species were L. parabrevis, L. nodensis, W. cibaria, L. buchneri, L. plantarum, L. paralimentarius, L. brevis, W. hellenica, L. sakei, and L. paucivorans. However, after 45 days of ensiling, Pseudomonas putida and Stenotrophomonas maltophilia sharply increased and remained predominant, followed by L. buchneri and L. acetotolerans.Relative abundances of bacteria (genus and species levels) are shown in  mixture . Further mixture . In pre-L. nodensis was the most abundant species, whereas L. parabrevis remained predominant in J silage, followed by L. nodensis. Furthermore, compared to other treatments, J silage had more W. cibaria and L. sakei after 5 days of ensiling. L. parabrevis gradually replaced L. nodensis as the dominant species in all silage samples after 15 days of ensiling, whereas compared to C silage, relative abundance of L. parabrevis was increased by 23.8, 11.7, 5.8, and 10.3% in J, LB, LP, and LPB silages, respectively. For the J silage, there were more W. cibaria, whereas L. paralimentarius had the highest abundance in LPB silage after 15 days of ensiling. However, Acinetobacter lwoffii and Acinetobacter sp. were observed in C silage at Days 5 and 15 of ensiling, respectively, and Enterobacterales was identified in LP silage after 15 days of ensiling  relative abundances of W. cibaria (relative to other inoculated silages) after 15 days of ensiling. Furthermore, inoculation with L. plantarum had no obvious effect on increasing relative abundance of L. plantarum at 5, 15, or 45 days of ensiling. Relative abundance of L. buchneri in silages not inoculated with this bacterium was very limited (<1%) over the entire ensiling period, whereas inoculation with L. buchneri strains increased its relative abundance, with a sharp increase in LB and LPB silages after 45 days of ensiling.The succession of inoculations in the silage environment is shown in L. buchneri inoculants increased abundances of global and overview maps compared to other treatments.A heatmap of KEGG metabolic pathways during the ensiling process is shown in L. buchneri and pre-fermented juice improved significantly, relative to C and LP silage, with LB, LPB, and J silages stable for > 384 h. The pH of aerobic stability was affected (P < 0.01) by inoculant, ensiling days, and their interaction  of LAB in three genera were present in pre-fermented juice, with W. cibaria (96.9%) being dominant. W. cibaria was identified in the pre-fermented juice prepared from lucerne. Preparation of pre-fermented juice involved culturing microbes on the crop surface. That 81.6% of microorganisms in pre-fermented juice were also present in fresh lucerne confirmed that epiphytic microorganisms affected the composition of the microbial community in pre-fermented juice. Furthermore, Weissella, Lactobacilli, and Cocci species were reported as major components in various crops  was needed to be efficacious  by inoculating with LB, LPB, or pre-fermented juice; the latter is a promising biological source of LAB for cleaner production to improve silage quality.In this study, four species of LAB from three genera were detected in pre-fermented juice, with PRJNA798538). https://www.ncbi.nlm.nih.gov/bioproject/PRJNA798538.Publicly available datasets were analyzed in this study. This data can be found here: The sequencing data were submitted to the NCBI Sequence Read Archive database (accession number: LM: conceptualization, methodology, and writing—review and editing. QW: resources and formal analysis. XC: investigation and data curation. HL: methodology and investigation. ZZ: validation and supervision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Oryza sativa L.); thus, the BR signaling pathway is a very important tool for breeders to improve the grain yield and quantity of rice. Contrary to the well-established BR signaling pathway in Arabidopsis, there are significant gaps in the rice BR signaling pathway, especially the regulation mechanism from OsBSK3 to OsPPKLs and OsGSKs. In this study, we report how OsBSK3 knockout mutants confer shorter and lighter grains and exhibit a typical BR-insensitive phenotype, suggesting OsBSK3 plays a positive role in BR signaling without genetic redundancy with homologs. Furthermore, OsBSK3 could physically interact with OsPPKL1 and OsGSK3, the downstream components in BR signaling, as a scaffold protein, and inhibit the phosphatase activity of OsPPKL1 on the dephosphorylation of OsGSK3. In addition, the genetic evidence showed OsBSK3 acts upstream of OsPPKL1 in regulating grain length and weight. Our results clarify the role of OsBSK3 and provide new insights into BR-signaling mechanisms, leading to potential new targets for the genetic improvement of rice.Brassinosteroids (BRs) are a crucial class of plant hormones that regulate many important agronomic traits in rice ( Oryza sativa L.) is an important food crop worldwide and increasing grain yield is a major goal of rice breeding. Grain size and shape  are major determining factors for grain weight and yield, and also affect the quality and market value of rice [Rice  and its co-receptor BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1), and the signal is transduced to the central transcriptional factors BRASSINAZOLE RESISTANT 1 (BZR1) and bri1-EMS-SUPPRESSOR 1 (BES1/BZR2) through a series of phosphorylating or dephosphorylating events involving BR-SIGNALING KINASEs (BSKs), BRI1-SUPPRESSOR 1 (BSU1) and BRASSINOSTEORID INSENSITIVE 2 (BIN2). Among them, BSKs play central positive roles in controlling BR signaling [Arabidopsis genes, such as OsBRI1, OsBAK1, OsGSK2 (the orthologs of BIN2) and OsBZR1, have been shown to play conserved roles of BR signaling in rice, suggesting that the BR-signaling pathway is conserved in rice and Arabidopsis [Brassinosteroids (BRs) play important roles in regulating grain size and shape. In addition, BRs as major growth-promoting hormones play crucial roles in regulating various biological processes, including plant height, leaf angle, grain size and weight, suggesting that the manipulation of BR biosynthesis or signaling may provide promising strategies for improving rice yield ,5. BR-siignaling ,7,8,9. Ibidopsis ,12,13,14Arabidopsis. For example, GRAIN LENGTH 2 (GL2) encodes GROWTH-REGULATING FACTOR 4 (OsGRF4) and affects grain size and rice yield though BR signaling. The transcription activation activity of OsGRF4 could be inhibited by OsGSK2, the central negative regulator of rice BR signaling [GW5 encodes a calmodulin-binding protein, which is a major QTL affecting grain width and yield. GW5 physically interacts with OsGSK2 and represses its kinase activity, resulting in the accumulation of dephosphorylated OsBZR1 and DLT proteins in the nucleus to mediate growth responses (including grain width and weight) [qGL3 encodes a putative protein phosphatase with Kelch-like repeat domains (OsPPKL1), an ortholog of AtBSU1. However, OsPPKL1 plays as a negative role in BR signaling, in contrast to AtBSU1. qGL39311 dephosphorylates and stabilizes OsGSK3, which suppresses BR signaling, resulting in decreased grain length [OsBSK3, the ortholog of AtBSK3, belongs to a sub-family of receptor-like cytoplasmic kinases (RLCKs) (RLCK-XII) and is reported to play a conserved role in regulating BR signaling [Arabidopsis bri1-SUPPRESSOR1 (AtBSU1). OsBSK3 RNAi co-suppression lines (the expression of OsBSK1~4 is suppressed) showed smaller and lighter seeds than wild type (WT) plants [OsBSK3 regulates downstream genes, such as OsPPKLs and OsGSKs, remained unclear.However, some unique BR-signaling components have been identified in rice that display different regulatory mechanisms from those in ignaling . GW5 enc weight) . The majn length . Althougignaling . OsBSK3 ) plants . HoweverOsBSK3 acts upstream of OsPPKL1 in regulating grain length and weight in genetics. Our results provide new insights into BR-signaling mechanisms and may help improve grain yield and quantity in rice.In this study, we found that OsBSK3 positively controls grain size and weight without genetic redundancy with its homologs. Furthermore, OsBSK3 could physically interact with OsPPKL1 and OsGSK3, the downstream components in BR signaling, and inhibit the phosphatase activity of OsPPKL1 on the dephosphorylation of OsGSK3. Moreover, we demonstrate Arabidopsis. The phylogenetic tree revealed that OsBSK3 is evolutionarily related to AtBSK3, AtBSK4, AtBSK7 and AtBSK8, members that have been reported to be involved in BR signaling in Arabidopsis  background. Two independent lines (KO-1 and KO-2) with a 1 bp insertion and a 14 bp deletion, respectively, in the first exon of OsBSK3, resulting in different reading frame shifts, were selected and used for phenotypic investigation  treatment compared to the WT  assay to test the interaction between OsBSK3 and OsPPKL1. The result showed that the OsBSK3 protein could strongly interact with OsPPKL1  and OsPPKL1 fused to the C-terminal half of Venus (CVen) showed a strong fluorescent signal, indicating that OsBSK3 interacted with OsPPKL1  and the OsPPKL1&OsBSK3 (OsPPKL1&OsBSK3-KO) double mutant, resulting in different reading frame shifts and early termination, respectively  and displayed phenotypes similar to the WT line  [OsBSK3 has been reported to promote BR signaling in rice, but the precise molecular mechanism is unknown [OsBSK3 plays a positive role in BR signaling without genetic redundancy with its homologs. Therefore, the functional study of OsBSK3 is of great significance for unraveling the regulatory mechanisms of BSK family in BR signaling and the genetic improvement of rice.BRs are a classic of plant-specific steroid phytohormones, controlling major aspects of plant growth and development in both dicots and monocots. BSK family members have been shown to play crucial roles in regulating BR signaling, but the regulatory mechanisms are unclear. Many BSK members  have been reported to be involved in BR signaling in bidopsis ,21. Howemembers) , which p unknown . In thisArabidopsis and rice in recent years, the regulatory mechanisms of the BSK family remain largely unknown. The BSK family members lack the conserved regions for kinase activity, such as the conserved DFG motif and the classical GXGXXG motif (glycine-rich loop). Therefore, BSKs are regarded as a type of pseudo-kinases and may work as scaffolds, bridging multiple proteins to form complexes [Arabidopsis, AtBSK3 could be phosphorylated by AtBRI1 and promoted to interact with AtBSU1, which in turn inhibits the kinase activity of AtBIN2 through the dephosphorylation of AtBIN2 [Arabidopsis. One intriguing finding of this work is that OsBSK3 inhibited the phosphatase activity of OsPPKL1 on OsGSK3  was used for all transgenic experiments. The plants were grown in a paddy field at Shunyi Experimental Station of the Chinese Academy of Agricultural Sciences in Beijing during the natural growing seasons from 2019 to 2021.Nicotiana benthamiana L.) seeds were sown directly into soil and grown in a culture room at 26 °C (16 h light/8 h dark) for 1 month before being used.Tobacco (http://rice.plantbiology.msu.edu/ (accessed on 2 July 2021)) and NCBI ). Phylogenetic analysis was conducted by MEGA 7.0 using the Neighbor-Joining method with 1000 bootstrap replications based on the full-length amino acid sequences of BSKs homologs.All relative protein sequences were retrieved from (ubiquitin (LOC_Os03g10170) was used as an internal control. RT-qPCR assays were performed with an Applied Biosystems Q3 real-time PCR system, using the SYBR premix Ex TaqTM kit , according to the manufacturer’s recommended protocol. Each experiment was conducted with three biological replicates.Total RNA was extracted from the flag leaf of plants, using Trizol reagent . cDNA was synthesized using a QuantiTect Reverse Transcription Kit  and used as the template for quantitative real-time PCR (RT-qPCR) analysis. The rice OsBSK3 and OsPPKL1, specific sites for targeted mutagenesis were designed online (http://www.e-crisp.org/E-CRISPR/designcrispr.html (accessed on 20 May 2020)) [Agrobacterium tumefaciens strain EHA105 and used to transform to the callus of ZH11. The mutant plants were genotyped using PCR amplification and DNA sequencing. All transgenic lines were analyzed using the stable T2 progenies, unless otherwise indicated.To generate the CRISPR/Cas9 constructs for y 2020)) . The CRIy 2020)) . All conhttp://www.imagej.net (accessed on 15 January 2022)).The lamina inclination assays after BL treatment were performed as previously described . In brieOsBSK3, OsPPKL1 and OsGSK3 were cloned into the pGBKT7 at the EcoRI and BamHI restriction sites or pGADT7 at the EcoRI and BamHI restriction sites . The interaction assay was performed according to the manufacturer’s instructions (Clontech Yeast Protocols Handbook). Both the bait and prey constructs were co-transformed into the yeast strain AH109 and grown on SD/-Leu/-Trp medium, then further selected on SD/-Leu/-Trp/-His/-Ade medium.For GAL4-based yeast Y2H assays, the CDSs of OsBSK3, OsPPKL1, OsGSK3 and GW5 were cloned into the BiFC binary vector pDOE at the BamHI or PmlI restriction sites, respectively [N. benthamiana leaves for BiFC assays as previously described [For the BiFC assays, the CDSs of ectively . The exaescribed . The fluOsBSK3, OsPPKL1 and OsGSK3 were respectively cloned into the pCAMBIA-NLuc or pCAMBIA-CLuc vectors at the SalI and KpnI restriction sites. All constructs were confirmed through DNA sequencing and were introduced into the Agrobacterium tumefaciens strain EHA105. N. benthamiana leaves were co-injected with different pairs of nLUC and cLUC constructs as previously described [For the firefly luciferase complementation imaging (LCI) assays, the CDSs of escribed  and incuOsBSK3 and OsPPKL1 were cloned into the pMALc2x vector at the BamHI restriction site. The CDS of OsGSK3 was cloned into the pGEX-4T vector at the EcoRI restriction site. MBP- and GST-tagged recombinant proteins were purified according to the user’s manual, and were then incubated in kinase buffer (25 mM Tris-Hcl (pH 7.5), 10 mM MgCl2, 10 mM NaCl, 1.5 mM DTT, 100 μM ATP, 2× phosphatase inhibitor cocktail) at 30 °C for 1 h. Subsequently, the samples were analyzed using 10% SDS-polyacrylamide phos-tag gels as previously described [The CDS of All of the primer sequences are listed in


As a group, individuals with functional neurological disorder (FND) report an approximately 3-fold increase in adverse life experiences (ALEs) compared to healthy controls. In patients with FND, studies have identified a positive correlation between symptom severity and the magnitude of ALEs. While not all individuals with FND report ALEs, such findings raise the possibility of a trauma-subtype of FND.This study investigated if patients with FND, with or without probable post-traumatic stress disorder (PTSD) and/or significant childhood maltreatment, differed in their symptom severity and physical health.n = 34; functional movement disorder, n = 56). Participants completed self-report measures of symptom severity , physical health [Short Form Health Survey-36 ], childhood maltreatment [Childhood Trauma Questionnaire (CTQ)], and PTSD [PTSD Checklist-5 (PCL-5)]; a psychometric battery of other common predisposing vulnerabilities was also completed. To adjust for multiple comparisons, a Bonferroni correction was applied to all univariate analyses.Seventy-eight patients with FND were recruited  vs. those without probable PTSD (n = 43) had statistically significant increased scores on all symptom severity measures – as well as decreased physical health scores. In secondary post-hoc regression analyses, these findings remained significant adjusting for age, sex, race, college education, and: pathological dissociation; alexithymia; attachment styles; personality characteristics; resilience scores; functional seizures subtype; or moderate-to-severe childhood abuse and neglect scores; SOMS:CD and SDQ-20 findings also held adjusting for depression and anxiety scores. In a separate set of analyses, patients with FND and moderate-to-severe childhood abuse (n = 46) vs. those without moderate-to-severe childhood abuse (n = 32) showed statistically significant increased SDQ-20 and PHQ-15 scores; in post-hoc regressions, these findings held adjusting for demographic and other variables. Stratification by childhood neglect did not relate to symptom severity or physical health scores.Patients with FND and probable PTSD (This study provides support for a possible trauma-subtype of FND. Future research should investigate the neurobiological and treatment relevance of a FND trauma-subtype, as well as continuing to delineate clinical characteristics and mechanisms in individuals with FND that lack a history of ALEs. Functional neurological disorder (FND) is a prevalent neuropsychiatric condition characterized by distressing motor , sensory, and/or cognitive symptoms that are diagnosed based on positive features incongruent with other recognized pathology . Since eDespite the lack of diagnostic specificity, several independent cohort studies reported positive associations between the magnitude of previously experienced ALEs and functional neurological symptom severity –12. For A potential trauma subtype—representing a subgroup of patients with the same clinical diagnosis but with additional distinguishing features—is not unique to FND. Emerging data suggests that a range of neuropsychiatric conditions across the spectrum of traditionally conceptualized psychiatric and neurological conditions may potentially show characteristics of a clinically-salient “trauma subtype” , 18. ForIn this cohort study, we aimed to further investigate the possibility of a trauma-subtype of FND—with a focus on potential relationships to symptom severity and physical health. We stratified our mixed FND cohort based on the presence or absence of probable PTSD, and separately based on the presence or absence of endorsed childhood maltreatment (abuse and neglect considered separately) . Togethen = 56; including functional weakness (n = 24), tremor (n = 23), gait (n = 22), jerks (n = 2), dystonia (n = 4), and speech difficulties (n = 5)] and/or FND-seiz   were included. Exclusion criteria were illiteracy, history of mania or psychosis, active suicidality, and current illicit drug abuse or alcohol dependence. All participants provided written informed consent as approved by the institutional review board of Mass General Brigham. Note, psychometric findings from this cohort, unrelated to the present set of analyses, have been published elsewhere  based on “rule in” examination signs and semiological features were prospectively recruited for study participation following an outpatient evaluation at the Massachusetts General Hospital FND Unit from 2014 to 2020 see , 25. Incn = 30)] . FND dialsewhere , 27–29.The PTSD Checklist for DSM-5 (PCL-5) was used as one approach to stratify the FND cohort , 31. TheParticipants completed three symptom severity scales: the Somatoform Dissociation Questionnaire-20 (SDQ-20)—a 20-item questionnaire of functional neurological symptoms  in the past year with each item rated on a 5-point Likert-type scale from “Not at all” (scored 1) to “Extremely” (scored 5) , 35; thepost-hoc secondary analyses controlling for other relevant predisposing vulnerabilities, the following psychometric scales were also collected: Beck Depression Inventory-II (BDI)—a 21-item scale measuring depression symptom severity within the past 2 weeks, with each item scored from 0 to 3; the 20-items of the trait anxiety subscale of the Spielberger State-Trait Anxiety Inventory (STAI-trait), with each item scored in a 4-point scale ranging from “Almost Never” to “Almost Always”; the Dissociative Experiences Scale (DES)—a 28-item measure of dissociative experiences in daily life rated from 0 to 100%, with a cut-off mean value of ≥30% for pathological dissociation; Toronto Alexithymia Scale (TAS)—a 20-item measure of difficulty identifying/describing feelings and externally-oriented thinking, with each item scored on a 5-point Likert scale; Relationships Scales Questionnaire (RSQ)—a 30-item dimensional measure of adult attachment styles with each item scored on a 5-point Likert scale; NEO Five Factor Inventory-3 (NEO-FFI-3)—a 60-item measure characterizing the “Big Five” personality traits, with each item scored on a 5-point Likert-type scale from “Strongly Disagree” to “Strongly Agree”; and the Connor-Davidson Resilience Scale (CDRS)—a 25-item item scale accessing stress coping tendencies used in the past month, with each item scored on a 5-point Likert scale.To allow for n = 2) were omitted from analyses. We ran the Shapiro–Wilk test (command swilk) to determine the distribution of outcome measurements. A student t-test (command ttest) was used for normally distributed data and the Mann–Whitney Wilcoxon rank sum test (command ranksum) for non-parametric data, to investigate potential statistically significant differences in symptom severity  and/or SF36-physical health scores between our stratified FND subgroups: (1) FND-PTSD_high vs. FND-PTSD_low; (2) FND-Abuse_high vs. FND-Abuse_low; (3) FND-Neglect_high vs. FND-Neglect_low. A Bonferroni correction for multiple comparisons was applied to the alpha value of 0.05 across all univariate statistics; only findings that remained significant entered secondary (post-hoc) linear regression analyses.STATA-17 BE software  was used to run all statistical analyses, and missing data  and college graduate (yes/no), and other variables as follows: (1) only the previously mentioned demographic variables; (2) depression (BDI) and anxiety (STAI-trait) scores; (3) pathological dissociation (DES ≥ 30% yes/no); (4) TAS total score; (5) RSQ subscale scores for dismissing, fearful, preoccupied, and secure attachment styles; (6) NEO agreeableness, conscientiousness, extraversion, neuroticism, and openness personality trait scores; (7) CDRS total score; (8) FND-seiz (yes/no); (9) moderate-to-severe CTQ abuse and neglect scores (yes/no) in FND-PTSD_high analyses, or FND-PTSD_high and moderate-to-severe CTQ neglect (yes/no) if predicting FND-Abuse_high. For all regressions, variance inflation factor (estat vif) was used to look for multicollinearity amongst the independent variables.For all statistically significant univariate findings corrected for multiple comparisons, we conducted separate n = 33) vs. FND-PTSD_low (n = 43), FND-Abuse_high (n = 46) vs. FND-Abuse_low (n = 32), and FND-Neglect_high (n = 33) vs. FND-Neglect_low (n = 45).See The PHQ-15 and SF36-physical health scores were normally distributed, whereas the SOMS:CD and SDQ-20 scores were not. All symptom severity scores were significantly increased, and SF36-physical health scores significantly decreased, in patients with FND-PTSD_high vs. FND-PTSD_low. Additionally, patients with FND-Abuse_high vs. FND-Abuse_low showed statistically significant increases in SDQ-20 and PHQ-15 scores. There were no statistically significant differences in symptom severity or physical health scores in patients with FND-Neglect_high vs. FND-Neglect_low see .post-hoc linear regression analyses, FND-PTSD_high vs. FND-PTSD_low relationships to increased symptom severity and reduced SF36-physical health scores remained statistically significant adjusting for demographic variables and sequentially correcting for pathological dissociation; alexithymia; attachment styles; personality traits; resilience scores; FND-seiz; and for the other trauma scales. Only relationships between FND-PTSD_high and symptom severity as measured by the SDQ-20 and SOMS:CD also remained significant adjusting for depression and trait anxiety scores.In separate post-hoc linear regression analyses, FND-Abuse_high vs. FND-Abuse_low relationships to symptom severity  remained statistically significant adjusting serially for demographic and all other neuropsychiatric variables. There was no evidence of problematic collinearity across all regression analyses as measured by variance inflation factors. See post-hoc analyses.Additionally, in separate post-hoc analyses adjusting for other variables except for when controlling for depression and anxiety scores in SF36-physical health or PHQ-15 related analyses. Furthermore, stratification based on the presence of moderate-to-severe childhood abuse also showed statistically significant increases in symptom severity in the FND-Abuse_high subgroup—findings that held for all post-hoc analyses. Stratification per childhood neglect did not relate to either patient-reported symptom severity or physical health scores. This data lends support to the possibility of a clinically-significant trauma subtype of FND (based on high comorbid PTSD symptoms and/or high childhood abuse burden).Our cohort study found that the FND-PTSD_high subgroup reported increased symptom severity and decreased physical health compared to those in the FND-PTSD_low subgroup; this finding remained statistically significant in Consideration of a trauma subtype of FND is consistent with emerging themes in neurobiological research in this population. In gray matter quantitative analyses in patients with FND, childhood maltreatment and ALEs more broadly are linked to insula, amygdala, hippocampus, putamen, and cerebellum structural alterations , 39, 40.n = 186) vs. standardized medical care alone (n = 182)  , 50. Altn = 182) –53. For n = 182) ; other pn = 182) –58. Opern = 182) , 60.There are several gaps that remain with regards to the kind of data needed to adequately define a trauma subtype of FND. One of the most critical features to consider further is the nature of traumatic experiences and their impact on a given individual. In this study, the self-report of PTSD symptoms based on the PCL-5 with a cut-off indicative of probable PTSD drew significant results for the comparison of FND-PTSD_high vs. FND-PTSD_low in three symptom severity and one physical health measure. Alternatively, the CTQ abuse subscale measuring reported childhood sexual, physical and emotional abuse was used to divide individuals into FND-Abuse_high vs. FND-Abuse_low subgroups—with this stratification approach also leading to significant associations with two patient-reported symptom severity scores. The endorsement of active PTSD symptoms can be thought of as a proxy for the subjective current impact of past traumatic experiences. CTQ scores help quantify patient-reported childhood maltreatment regardless of concurrent PTSD symptoms. Additional work is needed to further understand if subjective ongoing relevance, subjective occurrence, or both are important factors in how to operationalize a trauma subtype of FND. Notably, large scale research has identified that the subjective occurrence of childhood maltreatment is a strong predictor of the development of psychopathology, irrespective of objective documentation of such experiences .This study has several limitations, including the lack of a DSM-5 structured psychiatric interview and no lifetime measure of ALE severity. We also do not have information on the specific timing of ALEs, and as such we are unable to comment on the potential influence of critical developmental periods in linking childhood abuse to later life FND symptom severity. Other nuanced trauma-related factors such as escape potential require continued inquiry , 62, as In conclusion, this study lends support to a possible trauma-subtype of FND–across the transdiagnostic spectrum of FND-movt and FND-seiz. Additional research is needed to replicate and expand upon the findings of this study, including investigating if neural mechanisms and clinical outcomes are the same or different in patients with FND with or without a history of childhood maltreatment (or concurrently active PTSD symptoms).dlperez@nmr.mgh.harvard.edu.The dataset presented in this article are not readily available because sharing of this data requires local IRB approval. However, on reasonable request by other researchers, anonymized raw data related to this research will be made available pending approval by the local IRB. Requests to access the dataset should be directed to DP, The studies involving human participants were reviewed and approved by Mass General Brigham IRB, Massachusetts General Hospital. The patients/participants provided their written informed consent to participate in this study.All authors were involved in all stages of preparing this manuscript, including the literature review, performance of statistical analyses, drafting and editing the manuscript, and approved the submitted version.


Transplantation of mesenchymal stem cells (MSCs) in the setting of cardiovascular disease, such as heart failure, cardiomyopathy and ischemic heart disease, has been associated with good clinical outcomes in several trials. A reduction in left ventricular remodeling, myocardial fibrosis and scar size, an improvement in endothelial dysfunction and prolonged cardiomyocytes survival were reported. The regenerative capacity, in addition to the pro-angiogenic, anti-apoptotic and anti-inflammatory effects represent the main target properties of these cells. Herein, we review the different preconditioning methods of MSCs  and the novel approaches  suggested to optimize the efficacy of MSC therapy. Several clinical trials have established the safety of mesenchymal stem cell (MSC) therapy and have shown promising results in the setting of cardiovascular disease over the past decades ,2. In isMSCs are undifferentiated, multipotent and self-renewable cells recognized for their potential of differentiation ,7 and paThe purpose of hypoxic preconditioning is to prolong the short survival time of grafted MSCs in the ischemic area, a major limitation of the therapeutic potential of stem cell therapy ,19,20. I2O2-related senescence by up-regulating the expression of VEGF, TGF-β and LDH [kBp65 [Numerous growth factors, drugs and pharmacological and chemical substances have been used for MSC preconditioning . For exa and LDH . The proH [kBp65 , apple eH [kBp65 , oxytociH [kBp65 , LL-37 bH [kBp65  and migrH [kBp65 . AlthougH [kBp65 , IL-1β bH [kBp65  and TGF-H [kBp65 . In addiH [kBp65  and dimeH [kBp65  pretreatH [kBp65 . Our resGenetic modification of MSCs up-regulates the expression of specific genes implicated in MSC migration, adhesion, survival and premature senescence . To begiExosomes are classified as extracellular vesicles that are continuously produced and released by various hematopoietic and non-hematopoietic cells ,105,106.Cell sheets and cell containing scaffolds represent the two forms of engineered cardiac patches . MultiplOverall, the therapeutic benefits of MSCs have been demonstrated in the treatment of ischemic cardiomyopathies ; howeverAs is known for most new therapies, the progression of MSC therapy has been hard, slow and punctuated by difficulties. The available evidence proves the safety of MSC transplantation, which represents a new, hopeful strategy for the management of cardiovascular disease, particularly ischemic and non-ischemic heart failure ,140,141.To conclude, transplantation of pre-conditioned MSCs results in better therapeutic efficacy in the setting of cardiovascular disease, especially with moderate hypoxia pre-conditioning. In parallel, the available novel techniques are able to overcome the limitations  of this regenerative medicine, promoting stronger cardiovascular outcomes. Starting translational engineered cardiac patch practice from pre-clinical trials in animal models to in-human trials may change our future management of heart failure.