Systemic sclerosis (SSc) is an autoimmune disease with high morbidity and mortality characterized by fibrosis of the skin and internal organs. Some studies have investigated the use of stem cells to treat SSc. Herein, a systematic review and meta-analysis was conducted to determine the efficacy and safety of mesenchymal stem cells (MSCs) in the treatment of SSc.PubMed, Embase, Cochrane Library, Web of Science, OVID, China National Knowledge Infrastructure and Wanfang databases were searched up to February 1, 2021. Literature screening, data extraction and quality assessment were conducted independently by two researchers in according to the inclusion and exclusion criteria. The discrepancies were resolved by a third researcher.P < 0.00001). 2. MSCs decreased the number of digital ulcer, mouth handicap scale, and visual analog scale of hand pain in SSc patients . 3. No statistical differences were detected in Raynaud's condition score and Cochin hand function scale score at 6 months of MSCs therapy (P = 0.5 and P = 0.62). 4. After 12 months of follow-up, MSCs improve carbon monoxide diffusing capacity and forced vital capacity of SSc patients (P < 0.05). 5. Overall, MSCs application was safe; a few cases exhibited swelling at the injection site, diarrhea and arthralgia, which had self-recovery, and no severe adverse events occurred in the included trials.A total of 9 studies encompassing 133 SSc patients were included in the study. Compared to the baseline after treatment with MSCs: 1. The modified Rodnan skin score (mRSS) was significantly reduced in patients with SSc (MSC therapy improves the degree of skin thickening, lung function, and mouth opening and relieves finger ulcers and pain in patients with SSc without severe adverse events. Thus, MSCs or MSCs combined with plasma and traditional medicine might be an effective and promising treatment of SSc patients.PROSPERO registration number: CRD42020200350 Systemic sclerosis (SSc), also known as scleroderma, is an autoimmune disease with high morbidity and mortality characterized by the fibrosis of skin and internal organs, including the heart, lungs, kidneys and digestive tract . The patStem cells play a key role in tissue homeostasis, repair, and regeneration. Also, autologous hematopoietic stem cell transplantation (AHSCT) has been included in the updated European League Against Rheumatism (EULAR) guidelines for treatment of rapidly progressing SSc . MesenchThere were no patient or public involved in this systematic review and meta-analysis. No patient was asked to advise on interpretation or write the results.This study was prospectively registered on PROSPERO (CRD42020200350) and performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines . The bodType of research: Clinical trials.Inclusion criteria: SSc adults treated with MSCs  were included, regardless of age, gender, disease duration and severity.Exclusion criteria were as follows: (1) Non-Chinese or English literatures; (2) Repeatedly published data; (3) Literature with incomplete data or lacked target indicators; (4) Non-clinical studies such as animal-based, review articles, case reports, conference reports, replies, patents or protocols.Type of participants and interventions:The effective outcome endpoints include at least one of the six aspects of the modified parameters of disease activity in SSc patients based on the EULAR scleroderma trial study group: recent skin changes, digital ulcer (DU), modified Rodnan skin score (mRSS), tendon friction rub, C-reactive protein (CRP), and diffusing capacity of the lung for carbon monoxide (DLco) in SSc patients . In addiSearch methods for identifying relevant trials.A comprehensive literature search was performed to identify the relevant publications in PubMed, Embase, Cochrane Library databases, Web of Science, OVID, China National Knowledge Infrastructure and Wanfang databases from their inception to February 1, 2021. The search strategies typically use a combination of terms from medical subject headings (MeSH) and free-text keywords. The English subject headings were “scleroderma, systemic” AND “mesenchymal stem cells”, combined with free words as follows:  AND . The Chinese subject headings are “硬皮病, 系统性”AND “间充质干细胞”, respectively. The combination of (系统性硬化症 OR 硬皮病 OR 局限性硬皮病 OR 弥漫性硬皮病 OR 重叠综合征 OR 进行性全身硬化症) AND(间充质干细胞移植 OR 间质干细胞 OR 脂肪间充质干细胞 OR 骨髓间充质干细胞 OR 脐带间充质干细胞) was searched as well. Manual search and other methods were supplemented to reduce the missed detection rate.The literature retrieved from each database was imported into the EndNote reference manager, and the duplicated articles were removed. Then, titles, abstracts and full texts were scrutinized to determine the eligible studies after excluding the irrelevant articles by two investigators (CJH/JL) independently. Any disagreements were resolved by a consensus. Any discrepancies were addressed by a third researcher (DM) who decided on the final results. In the case of unavailability of full-text, the data were obtained by contacting the original author through telephone or e-mail.Two investigators (CJH/CSX) independently extracted the data, including post-treatment outcomes, standard deviations, and the number of participants in each group. Then the data of the first author/published year, country, research type, number of cases , mean age, follow-up time, cell type and number of MSCs, injection method, and endpoint index were extracted. The risk of bias of the included randomized controlled trials (RCT) was evaluated using the Cochrane risk of bias assessment tool, and the risk of bias of non-RCT studies was evaluated using the Newcastle–Ottawa scale(NOS) , with reI2 value, indicating low, moderate and high heterogeneity with the thresholds of ≥ 25%, ≥ 50% and ≥ 75%, respectively. Typically, I2 > 50% indicates substantial heterogeneity. In this study, the fixed-effect model was applied for analysis if trials were homogeneous (I2 ≤ 50% and P > 0.1) and the random-effect model was applied for the meta-analysis if statistical heterogeneity was identified (I2 > 50% and P < 0.1); P < 0.05 indicated statistical significance. Sensitivity analyses were also analyzed to test the stability of the pooled results. Publication bias was evaluated by STATA software 15.1, using both Egger's linear regression method and Begg’s rank correlation test.The extracted data were pooled and analyzed using the Cochrane Collaboration Software Revman 5.3. For dichotomous data, pooled outcomes were presented as odds ratio (OR) and 95% confidence interval (CI), while continuous outcomes were expressed as a mean difference (MD) and 95% CI for analysis. Heterogeneity was statistically evaluated by According to the above search strategy, 678 articles were initially retrieved from 7 databases, and 343 were obtained after the removal of duplicates. Then the titles and abstracts were screened for potential eligibility, and 11 articles were considered for full-text review which met the inclusion criteria. Of these, 3 were independent studies of the same population with different follow-up durations –21. Fina6/Kg by intravenous or hypodermic injection.The characteristics of included studies are summarized in Table In this study, VAS, MHISS, mRSS, Raynaud’s condition score (RCS), CHFS, and DU were finally selected as effective outcome measures for quantitative analysis in 7 studies. In addition, 2 studies with different evaluation criteria, such as skin wrinkling sensation, skin elasticity, and postoperative satisfaction, and 1 study that assessed the changes in DLco in SSc patients before and after MSC treatment were systematically evaluated without quantitative analysis. Moreover, the safety of MSC treatment in SSc patients was evaluated by screening the original data about AEs in the included literatures , and the results indicated that the RCS of patients did not change significantly .As shown in Fig. P = 0.98, I2 = 0%) showed that the DU numbers in SSc patients were significantly reduced after 6 months of treatment with MSCs , significantly improving the hand ulcer pain in SSc patients; however, high heterogeneity was observed . A random-effects model that accounts for statistical heterogeneity between the studies and provides a more conservative estimate of the significance than a fixed-effects model was used. Sensitivity analysis was conducted by eliminating the studies sequentially. No significant changes were observed in combining the results, indicating that the outcomes were stable and reliable.As shown in Fig. P = 0.13, I2 = 57%), and results showed that ADSC treatment improved mouth opening, oral swallowing, masticatory function and maxillofacial morphology in patients at 12 months . Granel et al. [P = 0.62; Fig. CHSF is a questionnaire assessment for the extent of hand involvement in SSc patients, including 18 items: dressing, washing, other daily activities and working ability. , 33 The l et al.  showed t6 cells/kg) in combination with plasma exchange and cyclophosphamide and showed significantly improved DLco and FVC of the 3 patients after 12 months (P < 0.05), suggesting that MSCs ameliorate pulmonary function damage in SSc-ILD patients.Only 1 study compared the changes in DLco and forced vital capacity (FVC) before and after treatment with MSCs in SSc patients. The average age of 14 patients with diffuse SSc was 37.4 years, and 3 of them were complicated with ILD. Patients with SSc-ILD showed no improvement in pulmonary conditions after treatment with glucocorticoids and cyclophosphamide. However, they received a single intravenous infusion of UC-MSCs . No serious AEs occurred in all the patients with MSC therapy Table .P = 0.349) and Begg’s test (P = 0.806) showed that the effect of MSCs from different sources on mRSS in SSc patients was symmetrical, suggesting no publication bias  combined with conventional therapy significantly reduced mRSS in SSc patients at 6 and 12 weeks with improved nail-fold microcirculation. In addition, functional organ damage in patients with SSc is closely related to the degree of fibrosis. Skin involvement often leads to orofacial skin thickening, lip skin wrinkling, and maxillofacial morphological changes, and even limited mouth opening, hand tendon contracture with dysfunction in severe cases. Herein, we found that MSCs promote skin elasticity and tightness at 3 months, reduce VAS and DU numbers at 6 months, decrease MHISS in SSc patients, and improve pulmonary function (DLco and FVC) of SSc-ILD patients at 12 months compared to pretreatment.After repeated screening and checking, 9 self-controlled studies were included in this study, encompassing 133 SSc patients. The clinical application of mRSS is a vital technique for evaluating skin thickness in SSc patients. The current results demonstrated that MSCs improve mRSS and reduce the degree of skin thickening in patients with SSc, regardless of cell sources or follow-up periods. A remarkable decrease in mRSS in SSc patients was observed at 12 months of follow-up after treatment. Moreover, compared to traditional therapy , UC-MSCs  and tumor growth factor-beta (TGF-β) play a critical role in angiogenesis. MSCs differentiate into dermal stromal cells and epithelial cells, secreting multiple cytokines  to promote wound repair and angiogenesis . The stuFurthermore, the overall safety of SSc patients with MSC therapy was good without serious AEs. As mentioned above, 7/9 clinical studies reported AEs. A total of 16 AEs occurred in 114 SSc patients treated with MSCs (16/114), and the skin redness and swelling at the injection site were the most common AEs in patients, among which 1 case with local infection ameliorated after treatment with oral antibiotics, and 5 cases improved spontaneously. The other AEs, such as mild respiratory tract infection, diarrhea, or arthralgia, also recovered soon with symptomatic therapy. Another retrospective study recorded 1 case of tumor in 39 SSc patients with MSC therapy . CurrentIn summary, MSC therapy improves the degree of skin thickening, lung function, and mouth opening, as well as relieves finger ulcers and pains in patients with SSc without severe AEs. MSCs or MSC combined with plasma and traditional medicine might be an effective and promising alternative for the treatment of SSc patients, especially those with severe disease, rapid progression of the disease, or refractory to conventional therapies. Alternatively, MSCs also decrease the titer or levels of serum antinuclear antibody and anti-Scl-70 antibody in SSc patients , 29. HowNevertheless, the present study has some limitations. Firstly, there are a few RCTs on MSCs in SSc treatment. Since the included literature consisted of self-controlled studies, control group assessment was lacking, and clinical outcomes may be interfered with by the natural course of the disease. Secondly, only a few studies were included in the quantitative analysis. Publication bias may exist because the power of the funnel plot test is insufficient, and some studies with negative results could not be published. Thirdly, the unclear description of methods, hidden groups, and outcomes of the included literature may affect the final results. Finally, the period of MSC therapy and follow-up is insufficient due to the small numbers of patients, and data of long-term efficacy and safety with large sample sizes are essential.MSCs therapy improves the degree of skin thickening, lung function, and mouth opening and relieves finger ulcers and pain in patients with SSc without severe AEs. MSCs or MSCs combined with plasma and traditional medicine might be an effective and promising alternative for the treatment of SSc patients. However, the certainty of evidence ranged from moderate to very low.


It is generally assumed that selective serotonin reuptake inhibitors (SSRIs) induce antidepressant activity by inhibiting serotonin (5-HT) reuptake transporters, thus elevating synaptic 5-HT levels and, finally, ameliorates depression symptoms. New evidence indicates that SSRIs may also modulate other neurotransmitter systems by inhibiting neuronal nicotinic acetylcholine receptors (nAChRs), which are recognized as important in mood regulation. There is a clear and strong association between major depression and smoking, where depressed patients smoke twice as much as the normal population. However, SSRIs are not efficient for smoking cessation therapy. In patients with major depressive disorder, there is a lower availability of functional nAChRs, although their amount is not altered, which is possibly caused by higher endogenous ACh levels, which consequently induce nAChR desensitization. Other neurotransmitter systems have also emerged as possible targets for SSRIs. Studies on dorsal raphe nucleus serotoninergic neurons support the concept that SSRI-induced nAChR inhibition decreases the glutamatergic hyperstimulation observed in stress conditions, which compensates the excessive 5-HT overflow in these neurons and, consequently, ameliorates depression symptoms. At the molecular level, SSRIs inhibit different nAChR subtypes by noncompetitive mechanisms, including ion channel blockade and induction of receptor desensitization, whereas α9α10 nAChRs, which are peripherally expressed and not directly involved in depression, are inhibited by competitive mechanisms. According to the functional and structural results, SSRIs bind within the nAChR ion channel at high-affinity sites that are spread out between serine and valine rings. In conclusion, SSRI-induced inhibition of a variety of nAChRs expressed in different neurotransmitter systems widens the complexity by which these antidepressants may act clinically. Major depressive disorder (MDD), also known as clinical depression, is a neuropsychiatric disorder characterized by depressed mood, sleep and cognitive disturbances, anxiety, anhedonia or decreased interest in enjoyable activities, and suicidal thoughts, among other symptoms . AlthougPrevious studies have determined that the malfunctioning of certain brain regions could be one important aspect in the development of MDD . The mosSeveral hypotheses have been described and proposed in order to elucidate the etiology of MDD, but each one includes various neurotransmitter systems ,7. One oThe cholinergic–adrenergic hypothesis proposes that the underlying cause of depression is an imbalance between the content of acetylcholine (ACh) and NA, producing an overstimulation of the cholinergic system over the noradrenergic system . CholineMounting evidence supports the view that other neurotransmitter circuits and pathways are involved in the clinical activity of SSRIs, including those modulated by nicotinic ACh receptors (nAChRs). This review focuses on functional, preclinical, therapeutic, and structural studies that support the activity of SSRIs on different nAChR subtypes.1A autoreceptors, which regulate 5-HT levels in the synaptic cleft. A high endogenous concentration of 5-HT may be sufficient for inhibiting nAChRs imipramine (a known tricyclic antidepressant and noncompetitive antagonist (NCA) of nAChRs [3H]TCP (a known NCA of Torpedo nAChRs [Torpedo nAChRs in the resting (toxin-bound) and desensitized (agonist-bound) states was determined. The studied SSRIs completely inhibited the specific binding of both [3H]imipramine [3H]TCP [i) for desensitized nAChRs were generally higher than those in the resting state (3H]imipramine binding to α4β2 AChRs with higher affinity than with α3β4 (~5-fold) and α7 (~10-fold) nAChRs, respectively (i’s in µM) for α4β2: fluoxetine (1.0 ± 0.1) > citalopram (4.1 ± 0.3) > paroxetine (6.7 ± 0.9), and for α3β4: citalopram (1.8 ± 0.1) > fluoxetine (4.8 ± 0.5) > paroxetine (6.9 ± 0.6), respectively  to o nAChRs ) to Torpipramine  and [3H] [3H]TCP , respectng state . In the ectively . Compariectively . These rectively .Although SSRIs generally inhibit nAChRs in a noncompetitive manner, the intrinsic inhibitory mechanisms are less clear. nAChRs exist in three allosteric transitions between different conformational states: a resting state, an active open-channel state, and several desensitized states, each with a non-conducting ion channel ,112. Eve2+ entry imipramine binding to different nAChRs   and α9(−) or another α10(−) (complementary component), but not at the α9(+) and α10(−) interface . To studMounting evidence supports the idea that the therapeutic activity of many commonly used antidepressants, including SSRIs, is partially mediated through the inhibition of nAChRs, thus counteracting the overactivity of the cholinergic system, which leads to depressive symptoms. New data highlight the importance of nAChRs expressed in different neuronal pathways in the regulation of 5-HT-induced effects. DRN serotoninergic neurons have been studied with enough detail to ascribe a potential modulatory activity of nAChRs, especially under stress/depression conditions. The evidence that SSRIs also inhibit a variety of nAChRs expressed in different neurotransmitter systems increases the complexity by which these antidepressants may act clinically. Although “nicotinic modulation” of depression has been indirectly recognized, the development of specific ligands, such as inverse agonists and partial agonists, of therapeutic value is still lacking.


Highermethanol concentrations suppress hydrate growth by in-lattice methanolincorporation, resulting in the formation of “defects”,increasing the energy of the nucleus. At low concentrations, on thecontrary, the detrimental effect of defects is more than compensatedfor by the beneficial contribution of CH This makes understandingtheir formation mechanism important to develop antiplugging strategies.Several hydrate-nucleation mechanisms have been proposed, and molecularsimulation and experiments go hand in hand in elucidation thereof:10 the Labile-cluster hypothesis (LCH)7 hasproven to be less probable, although the variant local-structuringhypothesis (LHS),10 “blob”hypothesis (BH),11 and cage-adsorptionhypothesis (CAH) are more promising.16sI Methanehydrate is the most widespread clathrate type existingin nature in the permafrost and continental-shelf regions and constitutesa possible significant energy resource.30 suggesting that special simulation techniques are necessary to studythe process in detail. Lauricella et al. have examined free-energylandscapes for hydrate nucleation from metadynamics31 and nonequilibrium MD.23 Małolepszaet al. applied a generalized replica exchange algorithm for hydratenucleation.32 Bi et al. determined themethane-hydrate nucleation pathway and the free-energy profile bythe forward-flux sampling method.34 Recently,Arjun et al. performed transition path sampling simulations, revealingtwo possible nucleation channels, indicating that a low-temperaturetwo-step nucleation mechanism, consistent, e.g., with the BH, is replacedby a direct one-step crystallization process at higher temperatures.35Hydrate-nucleation occurs on the microsecond time scale,38 noting that substantialaccelerations are possible for relatively low dosages. Concerningmethanol, Kvamme has argued that the most efficient inhibitory effectarises when alcohol  acts as a solvent toward water.39 The possibility of low-dosage enhancement ofhydrate nucleation by methanol is less explored38 although itrepresents an intriguing possibility40 confirmedby Abay et al.41 and Amtawong et al.42 FTIR experiments indicate substantial hydrogenbonding of the methanol molecule with “cage walls”,43 constraining the convenient methanol orientationvis-à-vis incipient hydrate cages. These intracage interactionsoutstrip typical van der Waals forces and may well contribute to apparenthydrate-promoting effects.40 Simulationswere performed to investigate structural characteristics and hydrogenbonding of methanol at the solution–ice interface44 and for clathrate hydrates incorporating theadditive molecules.46 Finally, very recently, Su etal.47 found that methanol both enhancesand suppresses nucleation depending on the temperature of the system.At higher temperatures, above 250 K, methanol competes with waterto interact with methane prior to the formation of clathrate nuclei,thus disrupting the formation of water clathrates. Below this temperaturethreshold, methanol encourages water to occupy the space between methanemolecules, favoring clathrate formation. Anticipating our results,here we show that the effect of methanol is more complex and highlydepends on its concentration, with its ability to attract methaneor replace it altogether in the occupation of the clathrate cavitiespromoting nucleation when the concentration of the additive in thesolution is low.The effect of promoters or inhibitorson the underlying detailsof hydrate nucleation remains unclear. Peters and co-workers havedeveloped frameworks on how additives affect nucleation pathways ingeneral ,47 focuson the effect of temperature on the formation of methane clathratehydrate from a solution at a prescribed concentration of methanol,here we focus on the complementary problem of the effect of concentrationat a fixed temperature.We remark that while Su et al.48 The latter is very complicateddue to the complex water/methanol structure and how these structuresare affected by the presence of methane, which increases methane’slocal solubility at the water interface, promoting homogeneous-likehydrate nucleation in the thin layer of water close to the interfacewith CH4.49It is very important here to distinguishbetween methanol effectson heterogeneous hydrate nucleation in the interfacial region in contactwith a separate methane phase and nominally homogeneous hydrate nucleationinside water.52 This hints at the “double life” of methanol, promotingand inhibiting hydrate nucleation at low and high concentrations,although the mechanistic origins of such a bifurcated character aremanifestly unclear.Typical pipelinemethanol levels for flow assurance are ∼40wt %, and it was found that systems with insufficient methanol (<∼5wt %) experienced worse plugging than uninhibited systems.To address the open methanol-effect questionspresented above,the present study applies both nonequilibrium simulations and carefulexperimental measurements to determine more conclusively methanol-additiveeffects on methane-hydrate nucleation across a wide concentrationrange and to ascertain the microscopic mechanisms of the process.3OHCH = 0.22–2.24 × 10–3) were placed in a temperature-controlled methane-hydrate-formationpressure vessel at 2.9 °C, featuring chemically and heat-treatedmarine sand, and allowed to form methane hydrate exposed to ∼120bar methane gas, establishing the inferred 24 h hydrate yield 58 for samplingof the initial condition55 , and ref  for furt59 where molecules are represented by point particles interacting througha suitably tuned Stillinger–Weber force field.60 mW is impressive for thermodynamic properties but has artificiallyfast kinetics; however, bearing in mind its low computational cost,mW constitutes an excellent approach for qualitative hydrate-nucleationinsights. Methane–methane and water–methane interactionsare modeled by two-body terms.61 Consistentwith the other species, methanol is represented as a two-point particle,with united-atom methane constrained by SHAKE62 to a mW water, approximating CH3-OH crudely. Surprisingly,this simplistic and computationally efficient model quantitativelyreproduces the all-atom characteristics of liquid water/methanol solutionsand methane clathrate hydrates incorporating CH3OH in thecrystal structure (see the Supporting Information). Considering that a single nucleation event with all-atom potentialrequires ∼1 ms of simulations,25 the force model used in this work allows for a good compromise betweenthe feasibility of a rigorous statistical analysis of the nucleationprocess by D-NEMD and the accuracy of the computational atomisticmodel.Forwater, we use the “mW” model,4CH, were considered:0.038, 0.044, 0.052, and 0.058, labeled A, B, C, and D, respectively.We added three different methanol quantities to A–D, χ3OHCH of 0, 4, 8, and 16 × 10–3, leading to 16 systems, with 40–80 independent trajectorieseach .The computational sample consisted of a two-phase orthorhombicsimulation box, with methane reservoirs providing the gas to the solutionin the box’s central part. Four values of the solvated-methanemole fraction, χ44 The nucleation enhancementreduces at higher concentrations, lending further direct evidenceof methanol’s concentration-dependent promotion-inhibitiondichotomy.52 χ3OHCH < 0.35 ×10–3 serves to promote hydrate formation, whichdeclines as the methanol concentration increases. There is a reversetrend in the 40–50 mM range (χ3OHCH < 0.7–0.9 × 10–3), where additionalmethanol increases the hydrate yield; however, this trend changesto a more modest promotion vis-à-vis the no-methanol case aboveχ3OHCH ∼ 0.9 × 10–3, which is consistent with previous works. Indeed, three distinctrégimes of pressure drop were observed during hydrate formation, analogous to literatureresults,52 interpreted by the “shrinking-core” model.52 In the present work, each stage’s durationis plotted in Figure S6 for different methanolconcentrations: the first lasts roughly 45 min for all concentrationvalues. Stage II is considered crucial, while there is a direct relationbetween hydrate-formation yield and duration; stage III correlatesinversely with yield.In broad terms, 4CH = 0.044–0.058,with negligible induction time: at high concentrations, the systemis essentially already a supercritical blob readily evolving towardthe corresponding clathrate. The methanol-concentration effects onhydrate-formation kinetics for these cases—whether it inhibitsor enhances nucleation—are irrelevant. Thus, we focus hereprimarily on χ4CH = 0.038, where we foundmeaningful and important methanol effects.In the simulations, it was observed thathydrate nucleation occurredvery quickly with χncages(t(n) = inf{t > t0|ncages(Γ(t)) > n}, i.e., the first time the nucleus consistsof more than n cages. Nucleation rates can be evaluatedusing the mean first-passage time τ(n) (MFPT),23 the average value of t(n) over independent realizations of nucleation. If the nucleationbarrier is sufficiently high, τ(n) is givenaccurately by234 diffusion-limited clathrateformation. Here, τj is the nucleationtime, b is related to the Zeldovich factor (Z = b/√π), n* is the critical size, H(•) is the Heavysidestep function, and c = 1/νg is the inverse of the formation rate (vg = ∂n/∂τ). We report,in n for simulations at χ4CH = 0.038 for χ3OHCH = 0, 0.008,and 0.024; the intermediate concentration (χ3OHCH = 0.016) shows an overlap with the pure-methane case . Therefore, consistentwith the experiment in 3OHCH of 0.024 inhibits hydrate-formation kinetics, while0.008 promotes it .To analyze simulations,we follow the total number of completehydrate cages ncages,24,31 alAlthough D-NEMD and experiments have been conducted at differenttemperatures and pressures, with more aggressive temperature and pressuredriving forces needed in MD for ostensibly-homogeneous nucleation,as opposed to primarily heterogeneous nucleation in experiments, thepositive correlation between simulation and experiment methanol’shydrate-formation “double life” is rather striking.The different conditions, heterogeneous vs homogeneous, and pressureand temperature certainly call for a future confirmation of this positivecorrelation. Nevertheless, we believe that present results stimulateresearch and discussion on this theme.45Probing mechanistic hintsfor methanol’s “doublelife,” the key observation is that, under certain conditions,methanol molecules can become part of the hydrate lattice itself,forming hydrogen bonds with their OH groups, which is consistent withprevious experimental observations.12 and 51262 clathrate hydrate cages, which facilitates clathratenucleation, or itself plays the double role of the element of thehydrogen-bond network of the crystal nucleus and hydrophobic fillingof the cavities, also in this case facilitating nucleation. However,methanol incorporation in the clathrate structure, with its lowercoordination, introduces defects in the hydrogen-bonding structureof the growing crystal, thus increasing the energy of the nucleusof the prescribed number of cages. Indeed, at low concentrations,with methanol’s liquid-phase chemical potential lower, it canmore easily (or quickly) leave the growing clathrate nucleus, fosteringcrystallite growth. The fraction of methanol incorporated in the nucleusstructure grows with methanol concentration in the solution  and so does its hydrogen-bond networkdefect. This likely results in an increase in energy of the crystalliteand hence in an increase of the nucleation barrier. Indeed, MD analysisshows that the critical-nucleus size per se did not change markedlywith methanol concentration, while the nucleation rate does (cf. j (vide supra), shrinking—supportingthe idea of critical-nucleus stabilization, in keeping with the overallshrinking-core model.63 Given the smallsize of the clathrate critical nucleus, ∼2–4 cages (Thanks to its amphiphilic nature, methanol eitherhelps embed methanein a local environment consistent with 5solution S2 and sosolution S2 and soThisbroader “double life” interpretation cannotbe explained solely by methanol’s in-lattice incorporation;we admit that In closing, we present mechanistic and statistical insights intomethanol’s “double life” vis-à-vis hydrate-nucleationeffects, noting promotion at low concentrations and switching to industriallyfamiliar inhibition at higher concentrations and providing a mechanisticorigin of the phenomenon. Further molecular-simulation studies withfully atomistic force fields will be performed together with spectroscopicwork to confirm the relevance of in-lattice methanol incorporationfor clathrate-nucleation suppression/enhancement.


Introduction: It has not yet been possible to demonstrate the well-established increased bleeding risk related to antidepressants (ADs) with methods of pharmacovigilance as disproportionality analysis. As bleeding events related to ADs often occur under comedication with antithrombotics, ADs might not be considered causative of, but merely “linked” with the bleeding event. Therefore, we hypothesized that causality assessment of bleeding events related to ADs and the competitive impact of antithrombotics are factors contributing to the mentioned previous non-findings.Methods: We performed a case/non-case study based on data from VigiBase™ and calculated reporting odds ratios (RORs) for 25 ADs. We used individual case safety reports (ICSRs) that were differently categorized in the database regarding the type of association between drug and event. Furthermore, we investigated the competitive impact of antithrombotics by comparing RORs calculated with and without ICSRs related to antithrombotics.Results: Analysis of ICSRs that were categorized as causally associated with ADs resulted in detection of only 2 signals . Analysis of ICSRs irrespective of the type of association resulted in detection of 8 signals . In our analysis, consideration of ICSRs associated with antithrombotics as competitive substances did not have significant impact on signal detection.Conclusion: Categorization of the type of association between drug and event may affect quantitative signal detection toward reduced sensitivity. Causality assessment seems to significantly impact signal detection, probably particularly in rare, unknown, or clinically unremarkable adverse drug reactions. ADs appear to increase the bleeding risk considerably, even independent of antithrombotic comedication. TM, several biases need to be taken into account, for example, the notoriety bias (Antidepressants (ADs) are frequently used in the treatment of depression, anxiety disorders, and several other mental disorders –4. In adety bias , underreety bias , 19, andety bias , 21. Theety bias , 22. MorThe collection of data was done by a data search query at VigiBase™  using terms of the Medical Dictionary for Regulatory Activities (MedDRA). VigiBase™ contains over 20 million reports of individual case safety reports (ICSRs) . The seaIn the first analysis, the categorization chosen for the database query was “suspected/interacting” and, for the second analysis, “suspected/interacting/concomitant.” The categorization of a drug/event report as “suspected,” respectively, “interacting”  or “concomitant”  is made by the primary reporter of the ICSR. All drugs that are considered as “interacting” are also considered to be “suspected” drugs in the database . Drugs tWe performed a case/non-case study and calculated the reporting odds ratios (ROR) and the 95% confidence intervals (CI) for disproportionality analysis. For this type of study, all reports associated with the respective substance and the ADR of interest are defined as “cases” and all other reports in the database as “non-cases.” If an ADR related to a certain substance is reported more frequently in comparison with all other substances in the database, this is referred to as a signal. In this study, a signal is defined as a ROR >1 with a lower limit of the 95% CI >1. First, the RORs were calculated for both datasets (“suspected/interacting” and “suspected/interacting/concomitant”); in the second dataset (“suspected/interacting/concomitant”), the competitive impact of antithrombotics was evaluated as described in the following paragraph (2.4). In a previous analysis where only ICSRS categorized as “suspected/interacting” were analyzed, the effects of the competition bias, i.e., the comparison of RORs calculated with vs. without inclusion of ICSRs associated with antithrombotics, were found to be negligible . These substances were chosen because haemorrhages are considered as a so-called type A reaction related to antithrombotic agents, meaning that the ADR is a consequence of the pharmacological effect and therefore very common .TM, 990,119 of them were associated with the SMQ “Haemorrhage,” 257,500 with the SMQ “Gastrointestinal Haemorrhage,” and 10,908 with the PT “Upper gastrointestinal haemorrhage.” In the following, we will present the signals found for each of the investigated SMQs, respectively, PT, first in the analysis of ICSRs that were categorized as “suspected/interacting,” followed by “suspected/interacting/concomitant.” For the latter, signals detected after removal of ICSRs related to antithrombotics are additionally presented. The comparison between the ROR values for “suspected/interacting” vs. “suspected/interacting/concomitant” is presented in At the time of the database query, there were 18,709,028 ICSRs in VigiBaseIn the analysis of ICSRs categorized as “suspected/interacting” concerning the SMQ “haemorrhage” , no signal was found; using ICSRs categorized as “suspected/interacting/concomitant,” eight signals were detected: amitriptyline [ROR: 1.09 ], citalopram [ROR: 1.41 (1.38–1.45)], escitalopram (ROR: 1.35 (1.32–1.39), fluoxetine [ROR: 1.03 (1.01–1.05)], hypericum perforatum [ROR: 1.26 (1.10–1.45)], paroxetine [ROR: 1.05 (1.02–1.07)], sertraline [ROR: 1.22 (1.20–1.25)], and trazodone [ROR: 1.28 (1.25–1.32)]. After removal of ICSRs related to antithrombotics , nine signals were found (using ICSRs categorized as “suspected/interacting/concomitant”): Two new signals related to venlafaxine [ROR: 1.06 (1.02–1.09) and duloxetine [ROR: 1.07 (1.04–1.11)] were detected, while the signal related to paroxetine became non-significant [ROR 1.02 (0.99–1.05)]; the other signals persisted. Regarding the ROR values including the 95% CI for each substance for SMQ “Haemorrhage,” see In the analysis of ICSRs categorized as “suspected/interacting” regarding the SMQ “Gastrointestinal Haemorrhage” , no signals were found. Using ICSRs categorized as “suspected/interacting/concomitant,” eight signals were found: amitriptyline [ROR: 1.43 ], citalopram [ROR: 1.67 (1.60–1.74)], doxepin [ROR: 1.28 (1.14–1.43)], escitalopram [ROR: 1.37 (1.30–1.43)], mirtazapine [ROR: 1.41 (1.33–1.49)], paroxetine [ROR: 1.10 (1.05–1.15)], sertraline [ROR: 1.31 (1.26–1.36)], and trazodone [ROR: 1.56 (1.49–1.64)]. After removing reports associated with antithrombotics from the database  10 signals were found, from which four were new (using ICSRs categorized as “suspected/interacting/concomitant”): duloxetine [ROR: 1.13 (1.05–1.20)], fluoxetine [ROR: 1.07 (1.01–1.14)], nortriptyline [ROR: 1.18 (1.02–1.38)], and venlafaxine [ROR: 1.10 (1.03–1.17)]; the previously found signal for mirtazapine [ROR: 1.41 (1.33–1.49) before and 1.10 (1.00–1.21) after removal] and paroxetine [ROR: 1.10 (1.05–1.15) vs. 1.04 (0.98–1.11)] disappeared. For the ROR values including the 95% CI for each substance for the SMQ “Gastrointestinal Haemorrhage,” see In the analysis of ICSRs categorized as “suspected/interacting” regarding the PT “Upper gastrointestinal haemorrhage”  two signals were found: citalopram [ROR: 1.61 (1.15–2.25)] and escitalopram [ROR: 1.56 (1.06–2.29)]. Using ICSRs categorized as “suspected/interacting/concomitant,” eight signals were found: amitriptyline [ROR: 1.60 ], citalopram [ROR: 2.51 (2.14–2.93)], duloxetine [1.49 (1.23–1.81)], escitalopram [ROR: 2.23 (1.86–2.67)], mirtazapine [ROR: 3.05 (2.52–3.70)], paroxetine [ROR: 1.36 (1.11–1.66)], sertraline [ROR: 2.13 (1.84–2.45)], and trazodone [ROR: 2.29 (1.89–2.77)]. After removing reports associated with antithrombotics from the database  seven signals were found (using ICSRs categorized as “suspected/interacting/concomitant”); no new signals were detected; the previously found signals persisted with the exemption of paroxetine [ROR: 1.36 (1.11–1.66) vs. 1.13 (0.77–1.68)]. For the ROR values including the 95% CI for each substance for the PT “Upper gastrointestinal haemorrhage,” see In the present study, we evaluated whether the causality assessment/categorization of a drug/event report as causally linked (“suspected/interacting”) vs. any type of association (“suspected/interacting/concomitant”) has an impact on signal detection using an example of the bleeding risk related to ADs. Furthermore, we analyzed the risk of different types of bleeding events and the influence of the competition bias by evaluating the effect of antithrombotics as competitors.In a previous analysis, where we only analyzed causally linked drug/event-reports, we could not detect a significant number of signals, meaning that we were not able to demonstrate the well-known bleeding risk related to ADs . By contPrincipally, by considering a possible competition bias caused by antithrombotic agents, there was no major impact of the competition bias on disproportionality analysis in the present study concerning ADs and bleeding; in other studies, however, the competition bias was found to be relevant for the sensitivity in signal detection –22: PariOur results are in line with the current literature regarding antidepressants with serotonin reuptake inhibition and an associated increased risk of bleeding. The bleeding risk related to antidepressants has not yet been demonstrated on the basis of data from spontaneous reporting systems. However, there are several studies demonstrating an increased risk for haemorrhages related to ADs with different methods. In a population-based cohort study by Renoux et al., an association between an antidepressant's affinity for the serotonin transporter and the risk of intracranial haemorrhage was found; the risk was even further increased with oral anticoagulants as comedication . In a caTaking into account the presumably particularly increased risk of bleeding related to ADs in the gastrointestinal tract [especially the upper gastrointestinal tract ], we alsOne major limitation of the study is the common problem of underreporting in ADR databases , 35. In This is the first time the bleeding risk related to ADs could be demonstrated using pharmacovigilance data. In contrast to previous studies, drug/event reports were analyzed irrespective of the available information regarding causality. The results of the present study suggest that in disproportionality analysis, the restriction to association categories that imply a causal link between drug and event may impede the detection of signals, particularly regarding ADRs that are rare, unknown, non-severe , hard to recognize, or difficult to assign to a specific drug. Even after removal of all reports associated with antithrombotic agents, there were still signals related to ADs regarding an increased bleeding risk, indicating there is a relevant risk of bleeding related to ADs even when administered without antithrombotics.https://www.who-umc.org/vigibase/vigibase/.Publicly available datasets were analyzed in this study. This data can be found here: All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by RZ and MG. The first draft of the manuscript was written by RZ and MG. All authors commented on previous versions of the manuscript and read and approved the final manuscript.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.


Brassica napus is the third leading source of edible oil in the world. Genic male sterility (GMS) lines provide crucial material for harnessing heterosis for rapeseed. GMS lines have been used successfully for rapeseed hybrid production in China. MicroRNAs (miRNAs) play crucial regulatory roles in various plant growth, development, and stress response processes. However, reports on miRNAs that regulate the pollen development of GMS lines in B. napus are few.BnaA09g48720D, BnaA09g11120D, and BnaCnng51960D were cleaved by bna-miR398a-3p, bna-miR158-3p and bna-miR159a, respectively. Among the differentially expressed miRNAs, miR159 was chosen to analyze its function. Overexpression of bna-miR159 in Arabidopsis resulted in decreased seed setting rate, and shortened siliques, illustrating that miR159 may regulate the fertility and silique development in rapeseed.In this study, 12 small RNA and transcriptome libraries were constructed and sequenced for the flower buds from the fertile and sterile lines of two recessive GMS (RGMS) lines, namely, “6251AB” and “6284AB”. At the same time, 12 small RNA and transcriptome libraries were also constructed and sequenced for the flower buds from the fertile and sterile lines of two dominant GMS (DGMS) lines, namely, “4001AB” and “4006AB”. Based on the results, 46 known miRNAs, 27 novel miRNAs on the other arm of known pre-miRNAs, and 44 new conserved miRNAs were identified. Thirty-five pairs of novel miRNA-3p/miRNA-5p were found. Among all the identified miRNAs, fifteen differentially expressed miRNAs with over 1.5-fold change between flower buds of sterile and fertile lines were identified, including six differentially expressed miRNAs between “4001A” and “4001B”, two differentially expressed miRNAs between “4006A” and “4006B”, four differentially expressed miRNAs between “6251A” and “6251B”, and ten differentially expressed miRNAs between “6284A” and “6284B”. The correlation analysis of small RNA and transcriptome sequencing was conducted. And 257 candidate target genes were predicted for the 15 differentially expressed miRNAs. The results of 5′ modified RACE indicated that B. napus.Our findings provide an overview of miRNAs that are potentially involved in GMS and pollen development. New information on miRNAs and their related target genes are provided to exploit the GMS mechanism and reveal the miRNA networks in The online version contains supplementary material available at 10.1186/s12870-021-03306-w. Mf, Ms, and ms are three alleles at the same locus, with a relationship of Mf dominant over Ms and Ms over ms. The recessive allele is for normal fertility. The maintainers and restorers are easily screened. Thus, multiple-allele DGMS are widely used for hybrid rapeseed seed production through the construction of a three-line hybrid system [Male sterility is largely due to the abnormal development of either the sporophytic or gametophytic anther tissues. According to its origin or genetic analysis, male sterility can be divided into cytoplasmic male sterility (CMS) and genic male sterility (GMS) . CMS is d system , 6.BnMs3, BnMs4, and BnRf). In 2012, Dong et al. demonstrated a major modification of the sterility inheritance model in 9012A. The modified inheritance model indicated that the male sterility was essentially controlled by two loci (BnMs3 and BnRf). The previously designated BnMs4 locus was just one allele of BnRf; it was then designated as BnRfa, which was designated in addition to BnRfb  and BnRfc . The dominance relationship of the three alleles is in the following order: BnRfa > BnRfb > BnRfc. The BnRf allele-specific molecular markers were identified; these markers would simplify the breeding process involving this RGMS line [Additionally, the recessive GMS (RGMS) systems have another distinct advantage. In RGMS systems, most inbred lines can restore their fertility, so hybrids with strong heterosis are easily bred. Several RGMS lines have been successfully commercialized in China, including S45AB , 117AB , and 901GMS line .Brassica napus, 2n = 38, AACC), which has low erucic acid and glucosinolate contents, is the third leading source of edible oil worldwide. In recent years, some conserved and novel miRNAs associated with silique length [Oilseed rape . Meanwhile, 12 small RNA and transcriptome libraries were also constructed and sequenced for the flower buds from the fertile and sterile lines of two DGMS lines (“4001AB” and “4006AB”). The aims of this study were to identify known and potential novel miRNAs from the 24 libraries and to analyze the expression profiles of the miRNAs and their targets in relation to DGMS and RGMS during rapeseed microspore development. The results would provide a foundation for evaluating the important regulatory roles of miRNAs in pollen formation and GMS occurrence in rapeseed and other crops.https://dataview.ncbi.nlm.nih.gov/object/PRJNA743414?reviewer=t674c02cj415380e8oldre4s5a). After removing the low-quality reads and contaminated adapter sequences, the clean reads of the 24 sRNA libraries ranged from 19.77 to 41.61 million. The mapped reads were further annotated against the Pfam database and subsequently divided into rRNAs, tRNAs, snRNAs, snoRNAs, ta-siRNA, and others. The endogenous sRNAs were identified as known and novel miRNAs. The average sRNA lengths of the three biological replicates for each sample were calculated, which showed the length distribution patterns of the sRNAs being similar to one another. In general, the majority of the small RNAs ranged from 21 nt to 24 nt in size. The 24 nt small RNAs were the most dominant, followed by 21 nt small RNAs  and fertile (6251B and 6284B) lines of the RGMS lines. Meanwhile, the flower buds were also respectively collected from the sterile  and fertile (4001B and 4006B) lines of the DGMS lines. Three biological replicates were conducted for each of the eight kinds of samples. Thus, total of 24 sRNA libraries were constructed and deep-sequenced. The raw reads of the 24 sRNA libraries ranged from 20.58 to 42.68 million  belonging to 15 miRNA families were identified  were identified for bna-miR159b, and two sub-members (bna-miR408a.1 and bna-miR408a.2) were identified for bna-miR408a. This phenomenon suggests that some MIRNA genes might be produced through a replication event from one origin to another one, which results in more copies of the miRNA group. Two members were identified for bna-miR319 and bna-miR398 families. Except the above mentioned five miRNA families, the rest of 10 miRNA families had only one miRNA member | > 0.73). As a result, 6, 2, 4, and 10 differentially expressed miRNAs were obtained between the flower buds of “4001A” and “4001B”, “4006A” and “4006B”, “6251A” and “6251B”, and “6284A” and “6284B”, respectively  compared with that in “4006A”. MiR159 and miR827 were both up-regulated in “6284A” flower buds compared with that in “6284B”. miR398 was up-regulated in “6284B” flower buds compared with that in “6284A” . A total of 560 transcripts were predicted to be targets of the 15 miRNAs -based analysis was performed to predict miRNA target genes with default parameters and a maximum expectation value of 3.5 (Bn.A09.CSD1 (BnaA09g48720D) was cleaved by bna-miR398a-3p. Bn.A09.PPR (BnaA09g11120D) was cleaved by bna-miR158-3p. Bn.Cnn.MYB (BnaCnng51960D) was cleaved by bna-miR159a.To further demonstrate the potential target genes, 5′ modified RACE was performed using mixed samples from flower buds of the fertile lines (“6284B” and “4001B”). Three target genes were validated using 5′ modified RACE  were detected in root, stem, rosette leaf, stem leaf, flower, and silique through qRT-PCR. The expression level of mature miR159a was the highest in silique (5875-fold), relatively lower in stem, stem leaf, and flower compared with that in root. The expression levels of AtMYB33 and AtMYB65 were very low and almost undetectable in silique, whereas they were relatively high in stem, stem leaf, and flower compared with that in the root  and MIR159OE-2 (13.6-fold) compared with that in WT. Meanwhile, the transcripts of AtMYB33 and AtMYB65 were suppressed in MIR159OE-1 and MIR159OE-2, especially in MIR159OE-2, compared with that in WT , Bn.A09.PPR (BnaA09g11120D), and Bn.Cnn.MYB (BnaCnng51960D) were cleaved by bna-miR398a-3p, bna-miR158-3p, and bna-miR159a. Additionally, overexpression of bna-miR159 in Arabidopsis resulted in decreased seed setting rate, and shortened siliques, illustrating that miR159 may regulate rapeseed fertility and silique development. All the results in our study would provide valuable clues for exploring miRNA-mediated regulatory networks in fertility development of GMS lines in B. napus.A large number of miRNAs were identified during pollen development in the two DGMS and two RGMS lines by deep sequencing. These identified miRNAs included 27 novel miRNAs on the other arm of known pre-miRNAs, 44 new conserved miRNAs, and 35 pairs of novel miRNA-3p/miRNA-5p. Among all the identified miRNAs, 15 differentially expressed miRNAs with over 1.5-fold change between flower buds of male sterile and fertile lines were identified, including six differentially expressed miRNAs between “4001A” and “4001B”, two differentially expressed miRNAs between “4006A” and “4006B”, four differentially expressed miRNAs between “6251A” and “6251B”, and ten differentially expressed miRNAs between “6284A” and “6284B”. The qRT-PCR results of 5 differentially expressed miRNAs  were consistent with deep sequencing results. The association analysis of small RNA and transcriptome sequencing was conducted, and the analysis results indicated that 257 genes were predicted to be the candidate targets of 15 differentially expressed miRNAs. The results of 5′ modified RACE verified that B. napus. The “6251A” and “6284A” are male sterile lines and the “6251B” and “6284B” are the fertile lines. Their male sterility is controlled by two loci (BnMs3 and BnRf) [B. napus. The “4001A” and “4006A” are male sterile lines and the “4001B” and “4006B” are the fertile lines. Their male sterility is controlled by three alleles  at the same locus [“6251AB” and “6284AB” are two recessive genic male sterile (RGMS) lines of nd BnRf) . The orind BnRf)  and Sun nd BnRf) . “4001ABme locus . The aboTotal RNA for each kind of sample was extracted in three biological replicates using Trizol reagent . RNA samples with an OD260/OD280 ratio of 2.0 and a total content of more than 2 μg were qualified for small RNA library construction. Then, 24 sequencing libraries  were constructed using TruSeqSmall RNA Sample Preparation Kit  and then sequenced using Illumina Hiseq 2500/Miseq at Beijing Novogene Bioinformatics Technology Co. Ltd.B. napus genome sequence (http://brassicadb.agridata.cn/brad/) using Bowtie2 with no mismatches allowed, more details were described in Niu et al. [Clean reads were obtained by removing low-quality reads, N-containing fragments, and adapters. Then, the length of 18 to 30 nt clean reads were mapped to the u et al. . UnmappeB. napus NAT-siRNAs in PlantNATsDB to remove NAT-siRNAs. Then miREvo [B. napus. To reveal the differentially expressed miRNAs, the miRNAs expression was analyzed using the DESeq2 [The mapped small RNAs reads were aligned to known miRNAs in miRBase22.1. Modified software mirdeep2  were usen miREvo  and mirdn miREvo  were usee DESeq2 . The onlhttp://www.mirbase.org/). The matched sRNAs with no more than three mismatches were considered as candidate conserved miRNAs, while the unmatched sRNAs were considered as candidate novel miRNAs. In addition, Mfold software was used to predict the secondary structures of pre-miRNAs with the flanking sequences of the candidate small RNAs in the genome [The candidate sRNAs were mapped to all the known plant miRNA sequences from the miRBase database  and oligo (dT) primers were used for first-strand cDNA synthesis. For the qRT-PCR analysis of mature miRNAs, U6 was used as internal control gene, and the first cDNA was synthetized using a miRNA First Strand cDNA Synthesis kit (Stem-loop Method) (Sangon Biotech). The qRT-PCR reactions were performed in a MyiQ2 qRT-PCR detection system  using iQ SYBR Green supermix (Bio-Rad) [-ΔΔCt method [Total RNA was treated with DNase I  to remove residual genomic DNA. For the qRT-PCR analysis of Bio-Rad) . Each ext method . All theA mixture of flower buds from the fertile lines (“6284B” and “4001B”) was used for total RNA isolation. The 5′ modified RACE was performed using a FirstChoice™ RLM-RACE Kit . Total RNA was directly ligated to the 5’RACE oligo. The first-strand cDNA synthesis and the two rounds of PCR reactions were conducted following the manufacturer’s instructions. The PCR products containing the target gene bands were ligated into pGEM-T Easy Vector  for sequencing . The priB. napus were designated as pre-miR159a-C6 and pre-miR159a-A7, respectively (Additional file B. napus using gene specific primers with endonuclease cleavage sites Sma I and Sal I. Then the fragments were cloned into pCAMBIA1301 binary vector with CaMV 35S promoter. The two vectors were designated as p35S::MIR159a-C6 and p35S::MIR159a-A7, which were introduced into Agrobacterium tumefaciens strains GV3101 and further transformed into Arabidopsis by floral dip. The inflorescences were dipped in the Agrobacterium solution containing sucrose and Silwet-77 for 2 min. The infected plants were cultured for 48 h in the dark environment and then transferred to greenhouse [1 and T2 hygromycin-resistant seedlings were transplanted and grown in greenhouse. Seeds from each transgenic plant were harvested separately. The corresponding Arabidopsis transgenic plants were designated as MIR159OE-1 and MIR159OE-2. The transgenic and wild-type plants were cultivated in the greenhouse under the same environment. The phenotypes of T1 and T2 transgenic plants were observed and recorded. Their silique length data were collected from more than three plants for each transgenic line. More than 30 siliques were measured for each plant.Two precursor sequences of bna-miR159a, which were located in A7 and C6 chromosomes of eenhouse . The seeAdditional file 1: Table S1. Identification of known miRNAs. Table S2. Precursors of 35 novel miRNAs in Brassica napus. Table S3. Target genes were predicted by psRNATarget. Table S4. The primers were used for qRT-PCR and 5, modified RACE analysis. Table S5. The sequences used for constructing MIR159 over-expressed vectors.Additional file 2: Figure S1. The secondary structures of new conserved miRNAs and novel miRNAs identified in Brassica napus.


Zinc plays a pivotal role in the function of cells and can induce apoptosis in various cancer cells, including Raji B lymphoma. However, the metabolic mechanism of Zn-induced apoptosis in Raji cells has not been explored. In this study, we performed global metabolic profiling using UPLC−Orbitrap−MS to assess the apoptosis of Raji cells induced by Zn ions released from ZnO nanorods. Multivariate analysis and database searches identified altered metabolites. Furthermore, the differences in the phosphorylation of 1380 proteins were also evaluated by Full Moon kinase array to discover the protein associated Zn−induced apoptosis. From the results, a prominent increase in glycerophosphocholine and fatty acids was observed after Zn ion treatment, but only arachidonic acid was shown to induce apoptosis. The kinase array revealed that the phosphorylation of p53, GTPase activation protein, CaMK2a, PPAR−γ, and PLA−2 was changed. From the pathway analysis, metabolic changes showed earlier onset than protein signaling, which were related to choline metabolism. LC−MS analysis was used to quantify the intracellular choline concentration, which decreased after Zn treatment, which may be related to the choline consumption required to produce choline-containing metabolites. Overall, we found that choline metabolism plays an important role in Zn-induced Raji cell apoptosis. Zn is an essential trace element that plays an important role in cell functioning as a nutrient, cofactor of enzymes, and intracellular mediator . The rolMetabolomics can be used to identify metabolites that can present the phenotype of a cell or an organism. The possibility of discovering metabolites that affect phenotypes and their applications in metabolomics has been suggested . Cell meZn ions are also essential for the action of many enzymes, including DNA and RNA polymerases, which are involved in DNA damage and repair ,12,13,14First, we confirmed the Zn ion−induced apoptosis of Raji cells via flow cytometry , and untThe PCA score plots indicated a clear distinction between the Zn ion-treated and control groups . The R2 p-values less than 0.05 and q-values for the false discovery rate (FDR) less than 0.05. Among these metabolites, the levels of the 59, 58, and 51 metabolites changed significantly after Zn ion treatment for 12, 18, and 24 h, respectively.Generally, the levels of amino acids and their analogs, organonitrogen and organooxygen compounds, fatty acyls, glycerophospholipids, nucleosides, and nucleotide classes showed opposite trends between the control and Zn ion−treated groups.Prominent changes appeared in the levels of glycerophospholipids and fatty acyls. The levels of GPC, AA, DHA, DPA, and adrenic acid increased after Zn ion treatment for 12 h by 1100-, 80-, 43-, 46-, and 37-fold, respectively . The incn−acetyl−dl−methionine, increased after 12 h of Zn ion treatment, but decreased at 18 h. On the contrary, all of the levels of threonic acid increased, but the level of n−acetyl−dl−methionine decreased 24 h after Zn ion treatment. In particular, the levels of fatty acyls, nucleotides, and nucleosides were markedly decreased 24 h after treatment.Most amino acids, except for threonic acid and We analyzed 1380 proteins through a Full Moon kinase array to investigate the effect of Zn ions on the protein level. To determine the phosphorylated changes of each protein, the phosphorylated ratio was calculated as the phosphorylated−form protein intensity/unphosphorylated−form protein intensity. The top 10 highest and lowest changed phosphorylated/unphosphorylated protein ratios at each time point are shown in a heat map . Consistl-glutamic acid, arachidonic acid, stearic acid, acetyl−l−carnitine, and uridine, were clearly changed after 12 h of Zn ion treatment  signaling is also involved in this metabolic pathway, which was highly associated with the response to Zn ion treatment with IPA analysis .From the pathway analysis, it was found that the metabolites involved in choline metabolism were increased by zinc ion treatment A, and thTo determine whether Zn ions affect the intracellular choline concentration, we quantified the choline concentration in cells using LC−MS/MS. The choline level in the cells was significantly decreased by Zn ion treatment for the first 24 h, but the choline level differences between the control and treated groups were smaller at 24 h than at 12 h B. The leWe found a significant increase in fatty acids and GPC at the same time that the concentration of choline decreased and thus predicted that this change would affect Zn ion-induced apoptosis. Therefore, we conducted cell survival experiments to test whether increasing fatty acyls and glycerophosphocholine (GPC) contributed to cancer cell death induced by Zn ions. Glycerophosphocholine (GPC), arachidonic acid (AA), docosahexaenoic acid (DHA), and docosapentaenoic acid (DPA), the levels of which were increased more than 50-fold by Zn ion treatment, were added to the cells.The results showed that only AA significantly induced cell death. The survival rate after AA treatment decreased to 43% at 12 h and to 63% at 24 h A in a dop < 0.05) in the 12 h of Zn ion treatment, and the concentrations were 0.12 ± 0.012 μM per cell (106 cells) in the control and 13.98 ± 2.475 μM per cell (106 cells) in the Zn 12 h treatment group. Considering this concentration, the concentration for the immunoblot assay was determined to be 10 μM.Since Zn ion−induced apoptosis proved to be caspase-dependent apoptosis , we condWe found that cleaved caspase −3, −9, and PARP were much more abundant in the AA-treated cells than in the control cells, and cleaved PARP was significantly upregulated 6 h after treatment C,D. ThesZn ions are required for DNA and RNA polymerase and tumor protein p53 to properly function, and are involved in repairing DNA damage and signals ,12,13,14In particular, the level of arachidonic acid (AA) increased after Zn ion treatment. The enzyme c−PLA2 releases AA for eicosanoid synthesis , and it The levels of DHA and phosphatidylcholine also increased after Zn ion treatment, and the pattern of change was similar to that of AA. The anticancer effects of DHA are known to be due to the induction of ROS and subsequent peroxidation of lipids ,32. PhosAlthough not as dramatic as the metabolic change of the lipids, a change in the level of amino acids (from approximately 1− to 5−fold) was also observed with Zn ion treatment. In particular, an increase in glutamic acid and a decrease in uridine were found to contribute to cell death in the IPA analysis . UridineProteins play important roles in cell apoptosis; therefore, we used the protein Full Moon Phospho−Explorer Antibody Array to determine how proteins change after Zn ion treatment. Previously, a decrease in phosphorylated p53 was shown to lead to the apoptosis of SMMC−7721 and HuH−7 cells, and it decreased after Zn treatments . PPAR−γ From the IPA analysis, the five canonical pathways most significantly changed by Zn ions were glioma signaling, endothelin−1 signaling, Fc epsilon RI signaling, PLC signaling, and NF−κB signaling pathways . PLC sigAccording to the KEGG pathway search, the metabolites changed by Zn ion treatment are commonly found in the choline metabolism in cancer; therefore, we hypothesized that Zn ion treatment can affect the choline metabolism in Raji cells. The increase in phosphocholine (PC) and total choline−containing compounds has been identified as a metabolic characteristic of tumors ,44.To address the association between Zn ions and choline, we quantified the concentration of choline in the cells. The results showed that the choline level decreased upon Zn ion treatment up to 24 h, but the choline level differences between the control and treated groups were smaller at 24 h than at 12 h. On the contrary, the levels of FAs and glycerophospholipids increased within 24 h after Zn ion treatment, and the increase was higher at 12 h than at 24 h. Therefore, we hypothesize that the increases in FAs and glycerophospholipids are the result of choline consumption.Finally, it was concluded that the metabolism of GPC and fatty acids was prominently changed in Zn ion−induced apoptosis. Metabolic changes were found to be initiated earlier than protein signaling, and cell apoptosis was proven to be closely related to the choline metabolism.Therefore, it is thought that the choline metabolism is important in Zn ion−induced Raji cell apoptosis, but other metabolic pathways may also be involved in cell death.31−18:1 PC standard was obtained from Avanti Polar Lipids, Inc. . A CCK−8 assay kit was obtained from Sigma Aldrich . For Western blotting, horseradish peroxidase−conjugated anti−rabbit (#7074), horseradish peroxidase−conjugated anti−mouse (#7076), and anti−cleaved caspase 9 (#31245) antibodies were purchased from Cell Signaling Technology . Anti−cleaved caspase 3 (PA5−23921) and anti−cleaved PARP (44−698G) were purchased from Thermo Scientific .The Raji cell lines were obtained from the American Type Culture Collection . All the cell culture materials, including fetal bovine serum (FBS), antibiotic-antimycotic and medium, were purchased from Gibco BRL . LC−MS−grade methanol was purchased from Burdick & Jackson . Ultrapure water (18.2 MΩ∙cm) was obtained using a Milli−Q apparatus from Millipore . Formic acid, reserpine, choline chloride, arachidonic acid (AA), glycerophosphocholine (GPC), cis−7, −10, −13, −16, and −19−docosapentaenoic acid (DPA), and cis−4, −7, −10, −13, −16, and −19−docosahexaenoic acid (DHA) were all purchased from Sigma Aldrich . Phospho Explorer antibody microarrays designed and manufactured by Full Moon Biosystems, Inc. , contain 1318 antibodies. The 16:0−dHexamethylenetetramine (HTMA) and 20 mM zinc nitrate were dissolved in 500 mL of distilled water using the hydrothermal growth method. ZnO rods were obtained in bulk (2 g) and dried in an oven at 30 °C for 24 h. The dried ZnO rods were suspended and sonicated in 500 mL of RPMI media with 10% FBS and 1% antibiotic−antimycotic for 4 h. ZnO rods were filtered, and the Zn ion concentration in the medium was measured by quantitative analysis of the elements using inductively coupled plasma atomic emission spectroscopy.2 at 37 °C [The Raji cells were cultured with RPMI−1640 containing 10% heat−inactivated fetal bovine serum and 1% antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin) in a humidified atmosphere of 5% COMetabolites were extracted from cells extracted as described in previous work . The sup–1 and 40 °C.Cellular metabolic profiling was performed using an Ultimate 3000 UHPLC system consisting of an autosampler and a column oven coupled to an LTQ Orbitrap Velos Pro mass spectrometer . An ACQUITY UPLC HSS T3 column  was used at flow rates of 0.4 mL minGradient elution was performed using mobile phase A (0.1% formic acid in distilled water) and mobile phase B (0.1% formic acid in methanol) . All samFor the Phospho Explorer array analysis, Raji cells were incubated under the same conditions as for the metabolomic analysis with Zn ions: at a concentration of 10 μg/mL and cultured for 10 min and 1, 6, 12, and 24 h. The culturing time was determined by considering that protein expression can change prior to a metabolic change . A totalThe data were processed using Compound Discoverer 2.1 software  for peak deconvolution and data normalization. For the statistical analysis, the data were normalized to internal standards and DNA concentrations of each sample. Metabolic differences between groups were investigated by multivariate statistical analysis using the principal component analysis (PCA) algorithm in EZinfo . The quality of the PCA model was determined by cross-validation of parameters R2 and Q2, representing the explained variance and the predictive capability of the model, respectively . Moleculhttp://www.hmdb.ca/ accessed on 15 January 2019), METLIN (http://metlin.scripps.edu/ accessed on 15 January 2019), ChemSpider (http://www.chemspider.com/ accessed on 22 January 2019), and KEGG (http://www.genome.jp/kegg/ accessed on 2 March 2019). Differences in the cellular levels of each metabolite in the heat map were statistically evaluated by Wilcoxon−Mann−Whitney tests using MetaboAnalyst  [t−test was used to test the altered metabolite features between the cells at different times after their treatment with Zn ions and the control. The Ingenuity Pathway Analysis (IPA) system (http://www.ingenuity.com/ accessed on 26 July 2019) was used to identify relevant pathways. A p-value of <0.05 was considered statistically significant: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001.The identification of metabolites in the analysis was putatively based on annotated compounds corresponding to level 1 or 2 of the metabolomics standard initiative (MSI) . The MS/ly 2019) . A StudeTo evaluate the metabolic change in the early phase of Zn ion treatment, the choline concentration in Zn ion-treated Raji cells was quantitatively analyzed. Metabolites in the control and 6, 12, and 24 h Zn ion−treated Raji cells were extracted as described for metabolic profiling, and 1−palmitoyl−d31−2−oleoyl−sn−glycero−3−phosphocholine (16:0–d31–18:1 PC) was used as the internal standard for the quantitative analysis of choline. The quantitative analysis was performed using an Exion LC chromatography system with triple-quadrupole mass spectrometry API4500 (AB Sciex) operated in positive ESI mode using multiple reaction monitoring. Chromatographic separation was performed using an Ultimate 3000 UHPLC system equipped with a UHPLC HILIC column . Analyst software (version 1.5.2)  was used for data acquisition and to control the equipment.6 cells).To clarify the AA concentration in Raji cells after Zn ion addition, we quantified the concentration of AA in the cell using Exion LC chromatography system with triple–quadrupole mass spectrometry API4500. The Raji cells were incubated with or without Zn ion treatment for 12 h. AA was extracted from Raji cell pellet as described in the metabolomics profiling. Chromatographic separation was assessed using ACQUITY UPLC BEH C18 column , and reserpine (2 mg/L) was used as the internal standard. The intracellular concentration was calculated as the concentration from the standard curve. Calculated concentration of AA was normalized by the cell number , glycerophosphocholine (GPC), cis −7, −10, −13, −16, an −19−docosapentaenoic acid (DPA) in dimethyl sulfoxide (DMSO) at concentrations of 10 and 20 µM for 12 and 24 h. After incubation at 37 °C for an additional 4 h, the absorbance was measured at 450 nm by a microplate reader.To determine the cell survival response to metabolites, we performed a Cell Counting KIT−8 (CCK−8) cell viability assay. After being seeded in a 96−well microplate at a density of 1.5 × 10After the Raji cells were treated with 10 µM of AA, they were harvested and lysed using cell lysis buffer  following the manufacturer’s instructions to confirm the apoptosis induced by AA. Proteins in the lysates were separated by SDS−PAGE and transferred to nitrocellulose membranes by an iBlot 2 system . After blocking in 5% BSA in TBS containing 0.5% Tween −20, the membrane was probed with primary antibodies and detected with horseradish-conjugated antibodies. Chemiluminescence was measured and quantified using a Bio−Rad ChemiDoc imaging system .5). Then, the cells were washed with cold PBS and resuspended in binding buffer containing PI and FITC−conjugated Annexin-V protein. The percentage of stained apoptotic cells was determined using flow cytometry (FACS) with a FACSCanto II cytometer. The percentage of the cells in the Annexin−V−FITC scatter plots was calculated for comparison.To identify the apoptotic Raji cells following Zn ion treatment, we performed an Annexin−V/propidium iodide (PI) staining assay using an EzWay Annexin−V−FITC apoptosis detection kit  in accordance with the manufacturer’s instructions. After treatment with 10 mg/L of Zn ions for 12, 18, and 24 h, the Raji cells were harvested (5 × 10


Improving the fertility of sheep is an important goal in sheep breeding as it greatly increases the productivity. Dolang sheep is a typical representative breed of lamb in Xinjiang and is the main local sheep breed and meat source in the region. To explore the genes associated with the initiation of puberty in Dolang sheep, the hypothalamic tissues of Dolang sheep prepubertal, pubertal, and postpubertal periods were collected for RNA-seq analysis on the Illumina platform, generating 64.08 Gb clean reads. A total of 575, 166, and 648 differentially expressed genes (DEGs) were detected in prepuberty_vs._puberty, postpuberty_vs._prepuberty, and postpuberty_vs._puberty analyses, respectively. Based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, the related genes involved in the initiation of puberty in Dolang sheep were mined. Ten genes that have direct or indirect functions in the initiation of puberty in Dolang sheep were screened using the GO and KEGG results. Additionally, quantitative real-time PCR was used to verify the reliability of the RNA-Seq data. This study provided a new approach for revealing the mechanism of puberty initiation in sheep and provided a theoretical basis and candidate genes for the breeding of early-pubertal sheep by molecular techniques, and at the same time, it is also beneficial for the protection, development, and utilization of the fine genetic resources of Xinjiang local sheep. GnRH neurons become less sensitive to the negative feedback effects of steroid hormones, while pulsatile GnRH secretion increases, stimulating gonadotropin secretion and ultimately leading to follicle development and ovulation  genes associated with proestrus by performing differential RNA-seq analysis of hypothalamic tissue in sows’ prepuberty, during puberty, and postpuberty. Dolang sheep is the most common sheep breed in the southern region of Xinjiang, known for their high litter rate, adaptability, and perennial heat . The regquencing . Similarquencing  identifiDolang sheep maintained in the Tarim University experimental station were used as the model in this study. The ewes were observed at 10 o ‘clock, 14 o ‘clock, and 18 o ‘clock every day. The criteria for judging puberty in ewes were mental restlessness, the tendency to walk, the acceptance of ram riding, and the presence of mucus in the vulva. All sheep were in good health. Hypothalamus samples were collected from ewes that were first found to be undergoing puberty, immediately frozen in liquid nitrogen, and stored at −80°C for further analysis. The hypothalami were further collected from prepubertal and postpubertal ewes maintained in the same manner. A total of nine sheep’s hypothalamic tissues were collected. Three biological replicates were performed for each period and analyzed.via exonuclease/polymerase activities. After adenylation of 3′ ends of DNA fragments, NEBNext adapter with a hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 240 bp in length, the library fragments were purified with the AMPure XP system . Then, 3 μl USER enzyme  was used with size-selected, adapter-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. Then, PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and index (X) primer. At last, PCR products were purified (AMPure XP system), and library quality was assessed using the Agilent Bioanalyzer 2100 system. Clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v4-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina platform (Biomarker Technologies), and paired-end reads were generated.The total RNA content from the hypothalami was extracted using TRIzol reagent (Beijing Kangwei Century Biotechnology Co.). The purity, concentration, and integrity of the RNA samples were tested by Nanodrop  and Agilent 2100 . A total of 1 μg RNA per sample was used as the input material for the RNA sample preparations. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using a random hexamer primer and M-MuLV reverse transcriptase. Second strand cDNA synthesis was subsequently performed using DNA polymerase I and RNase H. Remaining overhangs were converted into blunt ends In the present study, transcript libraries from Dolang sheep at the developmental stages of prepuberty, puberty, and postpuberty were constructed and assayed by high-throughput RNA-seq. Clean reads were obtained using the program Trimmomatic v0.32  by removTo assess the reliability of the tested samples, the degrees of variation among the three groups were analyzed using replicate scatter. It was finished by R package corrplot  and ggplp-value ≤ 0.05. The number of differentially expressed genes among the three groups was compared. Hierarchical clustering analysis was performed on the screened differentially expressed genes using R package heatmap.2  were identified using the DEseq2  method, eatmap.2 , and thevia alignment with multiple databases, including the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases.Gene function was annotated The soft clustering Mfuzzy function is based on the fuzzy k-means algorithm in e1071 package. R package Mfuzz was used to assign a gene to several clusters using soft clustering methods . This an2O, 7.5 μl PerfectStart Green qPCR SuperMix (2×), 1 μl cDNA, and 0.5 μl of each primer (10 μM). The thermal cycle parameters were as follows: 95°C for 15 s, 95°C for 15 s, 55°C for 15 s, and 68°C for 20 s for 40 cycles. Three technical replicates were performed for each sample. Actin was used as an internal reference. The relative expression levels of genes were calculated using the 2−△△CT method . Reverse transcription was performed using the reverse transcription kit (TaKaRa). The 15 μl PCR reaction mixture consisted of 5.5 μl ddHT method . SPSS 17This study was conducted in accordance with the specifications of the Ethics Committee of the Tarim University of Science and Technology.Pubertal features of Dolang sheep were observed . At the As shown in The clean reads in each library were then aligned to the sheep genome (Oar_v4.0). The proportion of clean reads mapped to the reference genome ranged from 84.60 to 89.71%, among which 88.74% were uniquely mapped. The percentage of reads mapped to sense strands ranged from 35.09 to 38.63% and those mapped to the antisense strands ranged from 39.35 to 41.95%.Gene expression levels were estimated by fragments per kilobase of transcript per million fragments mapped. $$FPKM = {cDNA fragments \over { mapped fragments (million) × transcript length (kb)}}$$. We used StringTie software to evaluate gene expression levels .Biological repeat correlation was calculated using R package of the corrplot . In the p-value ≤ 0.05 and fold change ≥ 1.5, including 490 upregulated and 85 downregulated genes. In the comparison of prepuberty and postpuberty, 166 genes exhibited a significant difference in their expression levels with the threshold of p-value ≤ 0.05, and fold change ≥ 1.5, including 96 upregulated and 70 downregulated genes. In the comparison of puberty and postpuberty, 648 genes exhibited a significant difference in their expression levels with the threshold of p-value ≤ 0.05 and log2 (fold change) ≥ 1.5, including 97 upregulated and 551 downregulated genes , “cellular response to gonadotropin stimulus” (GO: 0071371), “copulation” (GO: 0007620), “developmental process involved in reproduction” (GO: 0003006), “female pregnancy” (GO: 0007565), “estrogen receptor binding” (GO: 0030331), and “ovarian follicle development” (GO: 0001541). Some pathways related to puberty were made out, exempli gratia, “estrogen-signaling pathway” (ko04915), “oxytocin-signaling pathway” (ko04921), “GnRH-signaling pathway” (ko04912), and “progesterone-mediated oocyte maturation” (ko04914).GO terms associated with puberty were found, including StAR expression is upregulated and promotes cholesterol metabolism to pregnenolone in the ovarian steroidogenesis pathway. GIRK (G protein-gated inwardly rectifying potassium) showed higher expression levels in the estrogen-signaling pathway in prepuberty than in puberty. Upregulation of the SOHLH1 and GAMT genes was found in ovarian follicle development and reproduction, respectively. APC/C (anaphase-promoting complex/cyclosome), MYT1 (myelin transcription factor 1), and MAPK (mitogen-activated protein kinase) were upregulated during progesterone-mediated oocyte maturation. The prolactin signaling pathway members P38 and IRF-1 (interferon regulatory factor-1) were upregulated and downregulated, respectively.During the progression of prepuberty to puberty, Identified DEGs were annotated with 20 biological processes, 14 cellular components, and 10 molecular functions in the GO categories . The bin“copulation” (GO: 0007620), “ovarian follicle development” (GO: 0001541), “mating behavior” (GO: 0007617), “female pregnancy” (GO: 0007565), “fertilization” (GO: 0009566), “oocyte maturation” (GO: 0001556), and “cell differentiation involved in embryonic placenta development” (GO: 0060706) were included. Several pathways related to puberty, “oxytocin-signaling pathway” (ko04921) and “prolactin-signaling pathway” (ko04917) were further identified as differentially regulated.GO terms associated with puberty including p38MAPK leads to a reduction in the extent of its effect on gonadotropin gene expression and secretion. Upregulation of the DMC1 gene has been observed in ovarian follicle development. StAR was downregulated and returned to the same level as in prepuberty ewes. In the oxytocin-signaling pathway, protein kinase C (PKC) and GIRK are upregulated. p38MAPK (p38 mitogen-activated protein kinase) is downregulated in the GnRH-signaling pathway. In the prolactin-signaling pathway, p38 was downregulated, and TH and IRF-1 (interferon regulatory factor 1) were upregulated. In progesterone-mediated oocyte maturation, MAPK, Myt1, and APC/C were downregulated.As ewes progress from puberty to postpuberty, downregulation of These identified DEGs were annotated in 20 biological processes, 17 cellular components, and 13 molecular functions in the GO categories . The binGO terms associated with puberty were found, including “post-embryonic development” (GO: 0009791), “developmental process involved in reproduction” (GO: 0003006), “gonad development” (GO: 0008406), “copulation” (GO: 0007620), “fertilization” (GO: 0009566), “mating behavior” (GO: 0007617), “positive regulation of germinal center formation” (GO: 0002636), and “estrogen receptor binding” (GO: 0030331). Some pathways related to puberty were made out, exempli gratia, “ovarian steroidogenesis” (ko04913), “estrogen-signaling pathway” (ko04915), “oxytocin-signaling pathway” (ko04921), “progesterone-mediated oocyte maturation” (ko04914), and “GnRH-signaling pathway” (ko04912).PRL and TH were upregulated in the PRL signaling pathway. TTR was found to be downregulated in hormone activity entry.During the process of ewe development from prepuberty to postpuberty, calmodulin-dependent protein kinase (CaMKK), and soluble guanylyl cyclase (sGC) were found to be up- and downregulated in the oxytocin signaling pathway, respectively. SPIN1 gene in cluster3, DMC1 gene in cluster1, WISP1 genes in cluster2, and WNT2B gene in cluster4. All these genes play a direct or indirect role in puberty initiation .Based on these results, a number of genes associated with puberty were identified. The e system . SOHLH1 ogenesis . And SOHogenesis . DMC1 is meiosis . Loss of meiosis . The MACignaling . It was ignaling . Estrogeignaling . WNT2B mregnancy . Hatziroregnancy . SPIN1 reckpoint . CRH maycascades . TTR mayan sheep . WISP1 pchanisms . Co-exprp < 0.01). The presence of strong correlations indicates that the RNA-seq results were reliable  to verify by qRT-PCR and calculated the logarithm of the differential fold of gene expression . The correliable .NKB may be an important component of puberty initiation in sheep  associated with the initiation of sheep primiparity.GO classification of Dolang sheep transcriptome suggests that its properties are related to cellular components, biological processes, and molecular functions. Transcriptome analysis of Dolang sheep using the KEGG database identified the estrogen-signaling pathway, ovarian steroidogenesis, oxytocin-signaling pathway, progesterone-mediated oocyte maturation, prolactin-signaling pathway, and GnRH-signaling pathway. These pathways may be associated with the initiation of initial puberty and reproduction in Dolang sheep. Finally, we successfully identified nine genes (GAMT gene plays an important role in embryonic development (GAMT gene was upregulated in prepuberty_vs._puberty and downregulated in puberty_vs._postpuberty. This indicates that the GAMT gene starts to initiate expression during puberty in the preparation for embryonic development after mating. SOHLH1 is a transcriptional regulator that plays a role in the maintenance and survival of primordial ovarian follicles (SOHLH1 is upregulated in prepuberty_vs._puberty, possibly in preparation for sperm–egg cell binding. DMC1 plays a role in the initiation and progression of meiosis (DMC1 increased during the initiation and at the end of puberty in Dolang sheep, indicating that DMC1 may play a role in promoting oocyte meiosis during the initiation of puberty in Dolang sheep. The MACROD1 gene is also known as LRP16, and LRP16 plays a role in estrogen signaling (MACROD1 gene expression is increased, suggesting that MACROD1 may function during puberty initiation by influencing estrogenic signaling. WNT2B and WISP1 are co-expressed. Wnt2B and WISP1 are enriched in the Wnt-signaling pathway The Wnt-signaling pathway is expressed in granulosa cells, regulated by gonadotropins, and plays a role in follicle development, ovulation, and luteal formation.The elopment . In thisollicles . SOHLH1  meiosis . In thisignaling . In prepGAMT, SOHLH1, DMC1, MACROD1, WNT2B, SPIN1, CRH, TTR, and WISP1 genes were significantly different in their expression in the hypothalamus during different pubertal periods in Dolang sheep, suggesting that these genes may be key genes that directly or indirectly influence the initiation of puberty in Dolang sheep. These results provide a basic theoretical basis for further studies on the molecular mechanisms of pubertal initiation in Dolang sheep.In terms of mRNA expression,


The reamer irrigator aspirator (RIA) is a relatively recent device that is placed in the medullary canal of long bones to harvest a large volume of bone marrow, which is collected in a filtered canister. This study compares outcomes and complications of the RIA versus a traditional iliac crest bone graft (ICBG) for the treatment of bone defects.This meta-analysis was conducted according to the PRISMA guidelines. The Embase, Google Scholar, PubMed, and Scopus databases were accessed in June 2021. All clinical trials comparing the RIA and ICBG with a minimum of 6 months follow-up were included.P < 0.0001), fewer infections (P = 0.001), and a lower rate of adverse events (P < 0.0001). The ICBG group demonstrated a greater rate of bone union (P < 0.0001). There was no difference between groups in VAS (P = 0.09) and mean time to union (P = 0.06).Data from 4819 patients were collected. The RIA group demonstrated lower site pain (The current evidence supports the use of the RIA, given its low morbidity and short learning curve. Autologous bone grafting is a commonly performed procedure . ArthrodThis study compares the ICBG and RIA, seeking to demonstrate the noninferiority of RIA as a harvesting technique. The primary outcomes were the visual analogue scale (VAS) score and time to union. The secondary outcome includes the most common complications, such as donor site pain, fracture, infections, and hematoma/seroma.P (problem): long bones non-union, arthrodesis, osteomyelitis, maxillofacial surgery;I (intervention): autologous bone grafting;C (comparison): RIA vs ICBG;O (outcomes): PROMs, time to union, rate of union, complications;T (timing): ≥ 6 months follow-up.This systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses: the PRISMA guidelines . The PICTwo authors  independently performed the literature search in June 2021. PubMed and Google Scholar were accessed. Embase and Scopus were successively accessed to identify further articles. The following keywords were used in combination: “autologous,” “iliac,” “crest,” “bone,” “marrow,” “graft,” “reamer,” “irrigator,” “aspirator,” “posterior,” “anterior,” “ACBG,” “PCBG,” “ICBG,” “RIA,” “device,” “technique,” “long,” “bone,” “non-union,” “arthrodesis,” “osteomyelitis,” “PROMs,” “complications,” “morbidity,” “donor,” “site,” “surgery,” “harvesting,” “collection,” “medullary,” “canal,” and “invasiveness.” If the title and abstract matched the topic, the full-text article was accessed. The bibliographies of the full-text articles were screened for inclusion. Disagreements were resolved by a third author (**).All clinical studies comparing autologous crest bone grafting using the anterior or posterior harvesting technique with the RIA technique were accessed. Given the authors’ language capabilities, articles in English, German, Italian, French, and Spanish were eligible. Level I–IV evidence (according to the Oxford Centre of Evidence-Based Medicine) was considered. Only studies published in peer-reviewed journals were considered. Editorials, systematic reviews, meta-analyses, technical notes, narrative reviews, expert opinions, and letters were excluded. Animal, biomechanical, and cadaveric studies were also excluded. Only articles reporting a minimum of 6 months follow-up were included. Studies involving skeletally immature patients were not eligible. Only articles reporting quantitative data under the outcomes of interest were considered for inclusion.Two authors  independently performed data extraction. The following data were collected: generalities , demographic baseline , mean follow-up, mean BMI, indication for surgical intervention , and harvesting site. The following outcomes of interest were collected: visual analogue scale (VAS) and time to union (mean).Methodological quality assessment was performed by a single author (**) through the Coleman Methodology Score (CMS). The CMS is a reliable and validated tool to evaluate the methodological quality of systematic reviews and meta-analyses . This sc t-test, with values of P > 0.1 considered satisfactory. For the noncomparative studies included in the systematic review, the MD was used for continuous variables and the odds ratio (OR) for dichotomic data. The t-test and the χ2 test were performed, respectively, with values of P < 0.05 considered statistically significant. The confidence interval (CI) was set at 95%. Comparative studies were included in the meta-analyses. The meta-analyses were performed using Review Manager 5.3 software . For continuous data, the inverse variance method was used, with MD as the effect measure. For dichotomic data, the Mantel–Haenszel method was used, with OR as the effect measure. A fixed model analysis was used as default in all the comparisons. Heterogeneity was evaluated through the Higgins I2 test. I2 was interpreted according to the Cochrane Handbook for Systematic Reviews of Interventions (http://www.cochrane-handbook.org) as follows: 0–40%, poor heterogeneity; 30–60%, moderate heterogeneity; 50–90%, substantial heterogeneity; 75–100%, considerable heterogeneity. If I2 > 60%, we switched to a random model analysis. Values of P < 0.05 were considered statistically significant.The statistical analysis was performed by a single author (**) using the IBM SPSS software, version 25. Baseline comparability was assessed through the mean difference (MD) and the unpairedN = 400), a further 475 articles were found to be ineligible for the following reasons: study design (N = 365); language limitation (N = 17); short follow-up (N = 38); lacking quantitative data under the endpoints of interest (N = 49); cadaveric studies (N = 6). Finally, 40 comparative studies were included: one randomized controlled trial (RCT) and 10 prospective and 29 retrospective clinical studies. The literature search results are shown in Fig. The literature search resulted in 915 articles. After the removal of duplicates  was used to evaluate the risk of publication bias. The plot evidenced good symmetry, and all the referrals were located within the pyramid. Thus, the funnel plot indicated a low risk of publication bias Fig. .Fig. 2FuData from 4819 patients were collected, 1908 of whom were women. There was comparability between the two groups in terms of mean age, mean BMI, and mean harvest volume. Study generalities and patient demographics are shown in detail in Table P = 0.09) and mean time to union (P = 0.06) (Table There was no difference between the groups in terms of VAS (6) Table .Table 4CP < 0.0001), a lower incidence of infection , and a lower rate of adverse events . The ICBG group demonstrated a greater rate of bone union  compared to RIA. No difference was found in the fracture rate (P = 0.7) and the hematoma/seroma rate (P = 0.4). These results are shown in detail in Table The RIA group demonstrated lower site pain . Similarity was found in the length of the surgical intervention (P = 0.07), the transfusion rate (P = 1.0), the fracture rate (P = 1.0), the hematoma rate (P = 0.6), and the union rate (P = 0.4). The RIA demonstrated lower painful harvest site  and infection  rates. These results are shown in greater detail in Fig. Six studies that directly compared the RIA to ICBG were included in the meta-analysis , 23–26. According to the main findings of the present study, the RIA was associated with less morbidity than the ICBG. There was no difference in terms of VAS and mean time to union between the two groups. The RIA demonstrated a lower incidence of harvest site pain, with only 22 of 481 patients reporting this symptom, in contrast to the ICBG cohort, for which there were 674 cases of site pain in 1742 patients. Belthur et al. , 27–30. No difference was found between the groups in fracture or hematoma/seroma occurrence. The groups were similar in terms of surgery duration and transfusion rate, although the RIA is often reported in the literature to produce greater blood loss , 18, 26.Our study is not free of limitations. The retrospective design of most of the included studies is an important limitation. Unfortunately, only one study was a randomized clinical trial , which rCurrent evidence supports the use of the RIA, given its lower morbidity and shorter learning curve than ICBG. The RIA should become the new gold standard technique for bone marrow harvesting, but other clinical studies with long follow-ups are needed to prove it.