Following adjustment of clinically important variables, BARC 3&5 bleeds (OR 4.37) were still predictive of cumulative in-hospital and 30-day MACCE. In conclusion, major bleeding is an uncommon but potentially fatal PCI complication and was independently associated with greater MACCE rates. Efforts to mitigate the occurrence of bleeding, including radial access and judicious use of potent antiplatelet therapies, may ameliorate the risk of short-term adverse clinical outcomes.Bleeding severity in patients undergoing percutaneous coronary intervention (PCI), defined by the Bleeding Academic Research Consortium (BARC), portends adverse prognosis. We analysed data from 37,866 Australian patients undergoing PCI enrolled in the Victorian Cardiac Outcomes Registry (VCOR), and investigated the association between increasing BARC severity and in-hospital and 30-day major adverse cardiac and cerebrovascular events (MACCE) . Independent predictors associated with major bleeding (BARC groups 3&5), and MACCE were also assessed. There was a stepwise increase in in-hospital and 30-day MACCE with greater severity of bleeding. Independent predictors of bleeding included female sex (Odds Ratio (OR) 1.34), age (OR 1.02), fibrinolytic therapy (OR 1.77), femoral access (OR 1.51), and ticagrelor (OR 1.42), all significant at the  The cornerstone of treatment for patients presenting with acute coronary syndromes (ACS) is timely mechanical revascularisation with percutaneous coronary intervention (PCI) and potent antiplatelet pharmacotherapy. Bleeding is a recognised and important complication of PCI, and there is a significant body of evidence that associates peri-procedural  bleeding with poor clinical outcomes, including an increased risk of both short- and long-term mortality ,2,3. MajHowever, there is paucity of data in contemporary Australian practice pertinent to the clinical impact of PCI-related bleeding. Therefore, the primary objective of this study was to investigate the association between bleeding severity in PCI and short-term clinical outcomes in a large Australian multicentre registry of an all-comers population. This study used a standardised and robust model for defining bleeding, developed by the Bleeding Academic Research Consortium (BARC) . FurtherWe retrospectively analysed patient data from the Victorian Cardiac Outcomes Registry (VCOR) between January 2014 and December 2017. The VCOR is a state-wide population-based clinical quality registry that collects data related to PCI procedures and outcomes from all 30 public and private hospitals around Victoria. It is coordinated by Monash University in conjunction with the Department of Health and Human Services, and the Victorian Cardiac Clinical Network. The registry records on standardised case reports form PCI-related data, including patient demographics, procedural characteristics, and clinical outcomes and complications, with a 30-day follow-up period. Data are entered by trained hospital staff in real time as the patient progresses through a hospital admission, and data entry personnel are registered with VCOR. Participant data are prospectively entered into the VCOR unless they choose to ‘opt-out’. Regular auditing activities by the central registry ensures data integrity.The primary outcome was 30-day major adverse cardiac and cerebrovascular events (MACCE), a composite of clinical events occurring in-hospital and up to 30-days post discharge. MACCE was defined as all-cause mortality, new or recurrent myocardial infarction (MI) or stent thrombosis, target vessel revascularisation, or nonfatal stroke. Patients with missing 30-day MACCE data were excluded from the analysis.ACS was defined as a composite of ST-elevation myocardial infarction (STEMI), non-STEMI (NSTEMI), and unstable angina (chest pain at rest with unchanged cardiac biomarkers and ECG). Stable angina as an indication for PCI was defined as chest pain syndrome associated with exertion due to inducible myocardial ischaemia. Coronary lesion type was defined according to the American College of Cardiology (ACC) and the American Heart Association (AHA) classification as type A, type B1, type B2, and type C in order of increasing complexity .New renal impairment was defined as an increase of ≥25% or ≥44.2 mmol/L in serum creatinine up to 5 days after the index PCI, when compared to baseline. Cardiogenic shock was defined as a sustained (>30 min) episode of hypotension (systolic blood pressure (SBP) < 90 mmHg), or the use of vasopressors to maintain the SBP > 90 mmHg, together with evidence of end-organ hypoperfusion  and increased left ventricular filling pressures, such as pulmonary congestion. MI was defined as new elevation of cardiac biomarkers ≥ 20% compared to baseline levels, as well as one of the following: (1) ST-segment elevation distinct from the initial ischaemic event, (2) symptoms of myocardial ischaemia lasting ≥ 20 min, (3) development of new Q-waves in 2 or more contiguous leads, (4) angiographic evidence of flow limitation, and (5) evidence of new abnormalities in regional wall motion on imaging . Stroke In-hospital and outcomes up to 30 days were recorded, including rehospitalisation, readmission, MI, stent thrombosis, bleeding event, stroke, MACCE, and major adverse cardiac events (MACE), which was defined as MACCE minus stroke. These were verified from medical records, patients, and/or their primary care physician.The BARC bleeding criteria were divided into three categories: type 0 (no bleed), type 1&2 (‘minor’ bleed), and type 3&5  . A bleedp-values were calculated to demonstrate statistical significance between any two of the BARC groups. If significant (p < 0.05), post-hoc pairwise comparisons were conducted to evaluate which two BARC groups were different .Continuous variables are presented as mean ± standard deviation (SD) or median with interquartile range (IQR), and categorical variables are presented as frequencies and percentages. Differences between the 3 BARC groups were evaluated using one-way analysis of variance (ANOVA) or Kruskal–Wallis test for continuous variables, and a chi-square test for categorical variables. The independent effect of BARC-defined bleeding and other clinically relevant characteristics on cumulative in-hospital and 30-day MACCE (the primary outcome) was evaluated using a logistic regression model. Variables included in the multivariable analysis were the 3 BARC categories, characteristics that were clinically significant in the univariate analysis, as well as pre-defined variables that represented well-described predictors of bleeding, which included age, female sex, BMI, procedural creatinine, diabetes, peripheral vascular disease (PVD), out-of-hospital cardiogenic shock (OHCA), cardiogenic shock, fibrinolytic therapy, administration of glycoprotein IIb/IIIa inhibitors (GPI), and mode of vascular access ,15,16,17Yearly rates for mode of vascular access, BARC-defined bleeding, in-hospital MACCE, and cumulative in-hospital and 30-day MACCE from 2014 to 2017, among STEMI patients and ACS patients other than STEMI, were also shown for descriptive purposes.n = 34,555) experienced no bleeding (BARC 0), 7.9% (n = 3007) experienced a minor bleed (BARC 1&2), and 0.9% (n = 324) experienced a clinically significant major bleed (BARC 3&5). Notable baseline characteristics in this all-comers population included diabetes mellitus requiring medication prevalence of 22%, history of prior PCI (32.6%), history of prior CABG (7.7%), and moderate to severe heart failure (13.2%), defined by the New York Heart Association (NYHA) as left ventricular ejection fraction (LVEF) < 45%.Of 37,913 patients enrolled in the VCOR from January 2014 to December 2017, we analysed the data of 37,866 patients (n = 47 excluded for BARC 4 bleeding and missing 30-day MACCE data). The mean age was 66 years and 76.5% were male. Of 37,886 patients, 91.2% (p < 0.001). There were stepwise increases in BARC-defined bleeding among patients with worsening renal impairment (estimated glomerular filtration rate (eGFR) < 60%) with or without dialysis, those with moderate to severe LV systolic dysfunction, OHCA, cardiogenic shock, in-hospital pre-procedural cardiac arrest, or were administered GPI, or fibrinolytic therapy . In contrast, there were no significant differences between BARC groups in rates of patients with diabetes, patients on chronic oral anticoagulant therapy, and patients administered fibrinolytic therapy ≤ 24 h prior to PCI (p = not significant).Baseline patient characteristics of the three BARC groups are presented in p < 0.001). The BARC 3&5 group had higher rates of STEMI presentations, left main coronary artery (LMCA) involvement, coronary lesion type B2 and C, and the use of bare metal stents, and thrombus aspiration devices, procedural intubation, and extracorporeal mechanical support .Procedural characteristics of patients in the 3 BARC groups are presented in p < 0.001). For example, in-hospital MACCE occurred in 30.9% of patients who had BARC 3&5 bleeding, compared to 5.6% of patients who had BARC 1&2 bleeding and 2.7% of patients who had no bleeds . At 30-day follow-up, there was a graded increase in incidence of new heart failure, and 30-day MACE, MACCE, and mortality, with increasing severity of bleeding . There were also more rehospitalisations among patients with BARC 3&5 bleeding compared to patients who had BARC 1&2 bleeding or BARC 0 bleeding (p < 0.001).In-hospital outcomes and outcomes up to 30 days (events recorded from day of discharge to day 30 post discharge) within the three BARC groups are presented in p < 0.001). However, there were more patients in the BARC 3&5 cohort prescribed oral anticoagulants (p < 0.001).Medications prescribed at discharge are presented in p < 0.05).Using multivariable analysis, we identified predictors of cumulative in-hospital and 30-day MACCE. Cardiogenic shock was the strongest predictor : 11.61–16.86), followed by BARC 3&5 bleeding  . Other pp < 0.05) , female sex , fibrinolytic therapy , antiplatelet agents including GPI  and ticagrelor , severe NYHA heart failure (defined as LVEF < 30%) , cardiogenic shock , mechanical ventricular support , and procedural intubation   . Radial p < 0.001). Independent predictors of bleeding included female sex, age, fibrinolytic therapy, and ticagrelor use, while radial access was associated with a lower risk of bleeding .In this large, contemporary Australian multicentre registry of an all-comers population undergoing PCI, the major finding was a stepwise increase in in-hospital MACCE correlating with greater severity of bleeding: BARC 0 (2.7%), BARC 1&2 (5.6%), and BARC 3&5 (30.9%), as well as 30-day MACCE: BARC 0 (1.4%), BARC 1&2 (1.8%), and BARC 3&5 (3.1%). Additionally, there were increases in the length of hospital stay, and risk of rehospitalisation with BARC 3&5 bleeding. Following adjustment of clinically important variables, clinically significant BARC 3&5 bleeds remained predictive of cumulative in-hospital and 30-day MACCE  that evaluated the clinical impact of peri-procedural bleeding complications, incorporating various definitions of bleeding, including TIMI, GUSTO, STEEPLE, HORIZON-AMI, CRUSADE, BARC, and REPLACE-2 [n = 3,386,688) and the prospective ADAPT-DES trial (n = 8582), reported that 1.7% and 1% of patients experienced major bleeding respectively, which are congruent with our findings [Rates of major bleeding have been variably defined and variably reported among large, multicentre PCI studies ,19,20,21EPLACE-2 . In thisEPLACE-2 . It is rEPLACE-2  and infoEPLACE-2 . The mulRates of adverse in-hospital and 30-day outcomes in our study, especially mortality and MACCE, were similarly observed in analyses of other large registries including CathPCI ,24, the In our study, major bleeding was an independent predictor of cumulative in-hospital and 30-day MACCE (OR = 4.4) after adjusting for clinically important variables including age, sex, BMI, diabetes, PVD, pre-procedural creatinine, OHCA, cardiogenic shock, mode of vascular access, GPI, and fibrinolytic therapy. The independent adverse impact of PCI-related bleeding on clinical outcomes has been consistently reported in numerous studies ,19,20,26Fibrinolytic therapy and GPI use were also predictive of cumulative in-hospital and 30-day MACCE, independent of clinically important variables, including bleeding. In contemporary Australian practice, these agents are predominantly used in high-risk patients, which could explain the additive effect of GPI and fibrinolysis use on MACCE. Intravenous GPI is often administered in patients with high-risk clinical or angiographic characteristics , and fibrinolytic therapy is commonly used when there is delay in primary PCI, or if the patient is not at a PCI-capable centre . Howeverp = 0.0001) [Currently, aspirin and P2Y12 inhibitors  are the recommended antiplatelet treatment for patients during and after PCI, with studies showing significant reduction to 30-day MACE compared to aspirin alone ( 0.0001) . Patient 0.0001) . All the 0.0001) . A tailop < 0.001). Anaemia is known to independently predict adverse outcomes in patients undergoing PCI [The potential mechanisms by which major bleeding might increase mortality and MACCE are likely multifactorial. Some studies suggest that the frequent cessation of anti-thrombotic or antiplatelet therapy among patients who experience major bleeding, could subsequently increase their risk of ischaemic events ,26,33. Ioing PCI ,35. Thisoing PCI , or the oing PCI . There mIn our analysis, femoral vascular access predicted major bleeding , substantiating the results of numerous prior studies ,8. In paInterestingly, while the uptake of femoral access has significantly decreased since 2014, the rate of major bleeding, as well as in-hospital and 30-day outcomes, have not changed appreciably. Similar trends were noted in the MIG registry between 2005 and 2016 . A potenHowever, our study findings add to the body of literature emphasising the importance and adverse impact of bleeding on clinical outcomes. Any bleeding portends an adverse effect on outcome. These findings underscore the need for strategies to minimise bleeding, including adoption of radial access for PCI procedures and also meticulous care with prescription of antiplatelet and anticoagulant combination therapy among high-risk patients, such as those of older age, females, low BMI, and patients with severe heart failure.p-values presented in the univariate analysis should be interpreted with caution and regarded as hypothesis-generating because of the confounding effect of multiple testing. However, the data provide trends in patient and procedural characteristics across increasing severity of BARC bleeding. Secondly, there were likely additional confounding variables that were not measured or could not be measured which could impact clinical outcomes. Thirdly, BARC 0 and BARC 1&2 complications might have been underreported as these frequently did not require hospital-based intervention. Fourthly, patients who had CABG surgery without BARC 4 bleeds were not excluded, which might have affected 30-day MACCE outcomes. Fifthly, the timing of occurrence of any bleeding event in relation to development of in-hospital or 30-day MACCE was not individually verified or adjudicated given that VCOR is a state-wide database and the large number of clinical and bleeding events. Sixthly, VCOR also does not capture non-access site bleeding and severity of non-access site bleeds, which could have adversely impacted on outcomes. Finally, the follow-up duration of 30 days in our study was short, and longer-term follow-up may be necessary in order to ascertain significant differences in rates of adverse outcomes with contemporary procedural changes, including radial access and dynamic changes to antiplatelet and anticoagulant regimens.Our study results should be interpreted in the context of several limitations, including its retrospective design and observational nature. Firstly, due to the large number of variables being compared between the different BARC categories of bleeding, the results and Bleeding, especially major bleeding, is uncommon (~1%) in contemporary PCI practice. However, major bleeding is independently predictive of in-hospital and 30-day MACCE. With increasing severity of bleeding, there were also stepwise increases in in-hospital and 30-day mortality, length of hospital stay, and rehospitalisation. Efforts to mitigate bleeding and its consequences may improve patient morbidity and mortality.


Mesenchymal stem or stromal cells (MSCs) are nonhematopoietic postnatal stem cells with self-renewal, multipotent differentiation, and potent immunomodulatory and anti-inflammatory capabilities, thus playing an important role in tissue repair and regeneration. Numerous clinical and preclinical studies have demonstrated the potential application of MSCs in the treatment of tissue inflammation and immune diseases, including inflammatory skin diseases. Therefore, understanding the biological and immunological characteristics of MSCs is important to standardize and optimize MSC-based regenerative therapy. In this review, we highlight the mechanisms underlying MSC-mediated immunomodulation and tissue repair/regeneration and present the latest development of MSC-based clinical trials on cutaneous diseases. Mesenchymal stem cells (MSCs) represent a unique population of postnatal multipotent stem cells existing in a variety of adult, perinatal, and fetal tissues . Althougγ or during MSC differentiation [MSCs can be easily isolated from human donors and rapidly expanded in vitro without loss of their main biological properties . Up to dntiation . Therefoβ1 and PGE2 [α, G-CSF, and TGF-β [γ, and CXC chemokines [MSCs have potent immunomodulatory effects on various types of innate and adaptive immune cells . Among iand PGE2 . In addiand PGE2 , 30, leaand PGE2 –33 and iand PGE2 . With reand PGE2 –37 thround TGF-β , 38. Meaemokines , 40.β1, PGE2, and IDO [α, IL-1β, IFN-γ, CXCL9, TNF-α, CXCL10, and IL-17 [Among adaptive immune cells, MSCs could inhibit immune responses and proliferation of T cells either through direct cell-cell contact or through secretion of a large panel of soluble factors, such as HGF, TGF- and IDO . MSCs hand IL-17 , 42. On nd IL-17 , 43–45. nd IL-17 .β, PGE2, IDO, IL-10, TSG-6, and human leukocyte antigen-G5, are involved in MSC-mediated immunosuppressive and anti-inflammatory functions [γ and TNF-α, can stimulate the production of immunosuppressive mediators IDO and iNOS/NO in MSCs, which subsequently contribute to MSC-mediated immunosuppressive and anti-inflammatory functions [Accumulating evidence supports the notion that MSCs exert their therapeutic effects under various pathological settings through their paracrine secretion of a panel of trophic factors with a wide range of biological functions . A set ounctions . Of noteunctions . In addiunctions , keratinunctions , thus counctions , while sunctions , 50–54. μm), microvesicles , and exosomes (30-200 nm). EVs are widely distributed in body fluid and secreted in cell culture supernatants, which are rich in lipids, proteins, nucleic acids, and other components. Exosomes are released after fusion with the plasma membrane through forming multivesicular bodies, while MVs are released by directly shedding from the plasma membrane [Cells release extracellular vesicles (EVs) as one of the intercellular communication platforms. EVs could be mostly classified based on their diameter, including apoptotic bodies 1-5  μm, micrIn recent years, a growing body of evidence has shown that MSC-released EVs exhibit potent regulatory effects on various types of cells through transferring bioactive components to the recipient target cells. For example, MSC-EVs exert anti-inflammatory effects on inflammatory immune cells, such as M1 macrophages, DCs, and CD4+ Th1 and Th17 cells, through the intercellular transfer of immunoregulatory miRNAs and bioactive proteins, thus making them undergo phenotypic conversion into anti-inflammatory M2 macrophages, regulatory DCs, and Tregs. Additionally, MSC-EVs can promote tissue repair and regeneration through promoting the survival, proliferation, and regenerative potential of other types of resident cells. For instance, Wen et al.  showed tDue to the potent regenerative potentials and trophic paracrine effects, MSCs and their derivative products are emerging as promising therapeutics for a spectrum of diseases, including inflammatory skin disorders . ThroughVitiligo and alopecia areata (AA) are common chronic and recurrent autoimmune skin disorders characterized by white spots on the skin (vitiligo) and bald spots on the scalp (AA) because of selective destruction of melanocytes (MC) , 59, 60.β in the tumor microenvironment [Melanoma is the most malignant skin cancer, a process characterized by a linear transformation progression of normal melanocytes through various precursor lesions and ultimately to melanoma . Severalironment –65. Wangironment  reportedironment . Notwithironment . Most reironment .α/CXCR4 signaling axis [The successful use of hematopoietic cell transplantation (HCT) has previously been shown to treat pediatric patients with RDEB , 71, an ing axis  have beeing axis , 82. Curβ, increasing dermal thickness, and decreasing MMP and IL-6 expression [NCT01771679).Photoaging refers to skin aging associated with ultraviolet radiation (UVR) exposure . Jeong epression , 87–89. pression , 89, 90,Psoriasis is a T cell-mediated inflammatory autoimmune disease , charactDue to their potent immunosuppressive and anti-inflammatory properties, MSCs have been extensively explored as promising therapeutics for autoimmune skin diseases, such as dermatomyositis (DM) and alopecia areata (AA), and various systemic autoimmune/autoinflammatory diseases, such as systemic sclerosis (SSc), systemic lupus erythematosus (SLE), and graft versus host disease (GVHD) involved with severe skin manifestations (AA) –113. NumThe therapeutic effects of MSCs on chronic cutaneous ulcers, such as pressure ulcers, radiation skin ulcers, diabetic skin ulcers, and leprosy skin ulcers, have been evaluated in both preclinical studies and clinical trials . With reγ rather than increasing the expression of IL-10 [Atopic dermatitis (AD) is a typical abnormal T cell-mediated immune disorder characterized by a significant imbalance between Th2 and Th1/Th17, particularly in the early phase, whereas a mixed Th1/Th2 pattern appears in the chronic stage . MSCs frof IL-10 . On the of IL-10 . Anotherof IL-10 . Most reof IL-10 , 137. Inof IL-10 . CurrentWound healing is a complex process involving multiple layers of integrated interactions among various cell types and bioactive molecules, whereas any aberrant change in this process can lead to compromised wound healing, such as abnormal scar formation . In the β1, type I and type III collagens, and inflammatory responses; and increased expression of decorin [β3 showed obvious effects to reduce the formation of scar tissue in a rabbit skin wound model [Keloids are a skin disorder characterized by excessive collagen deposition into the extracellular matrix (ECM), while its pathogenesis remains largely unknown. Several lines of evidence have implied the potential role of MSCs with aberrant phenotypes and their special inflammatory niche in the keloid pathogenesis –166. Eve decorin –177. Rec decorin , 179. Ya decorin  showed tnd model . Most rend model , 183.To date, a growing body of preclinical and clinical studies has demonstrated the beneficial effect of MSC-based therapy for a wide spectrum of diseases, including various autoimmune and inflammatory skin disorders. However, there are several major challenges faced in the clinical translation of MSC-based regenerative therapies. One of the major challenges might be the large variations in the therapeutic efficacy of MSCs due to their heterogeneity caused by various intrinsic and extrinsic factors. Intrinsically, the different tissue origins, age, and health status (niche factors) can affect the property and function of MSCs. Extrinsically, the isolation, culture, and ex vivo expansion conditions can also affect the property and biological functions of MSCs . TherefoAccumulating evidence has shown that MSC-derived EVs exhibited potent immunomodulatory/anti-inflammatory and pleiotropic effects as the parental MSCs did. More recently, some preclinical and early clinical studies have shown that MSC-EVs displayed therapeutic effects on several disease models, including cutaneous diseases , 137. ThIn the last two decades, much progress has been made in delineating the molecular mechanisms of action of MSCs and their potential application in regenerative therapy for a wide spectrum of diseases, including various autoimmune and inflammatory skin disorders. To date, accumulating evidence supports the notion that MSCs exert their therapeutic effects under various pathological settings through multiple modes of functions mediated by their paracrine secretome containing a myriad of bioactive factors, including extracellular vesicles (EVs). However, much effort is still required to further investigate the specific cellular and molecular mechanisms by which MSCs of different tissue origins and their cell-free derivative products exert their unique therapeutic effects on certain cutaneous diseases.


Triticumaestivum and their response to plant viruses remain unknown. Here, we identified and characterized 49 T. aestivumHDACs (TaHDACs) at the whole-genome level. Based on phylogenetic analyses, TaHDACs could be divided into 5 clades, and their protein spatial structure was integral and conserved. Chromosomal location and synteny analyses showed that TaHDACs were widely distributed on wheat chromosomes, and gene duplication has accelerated the TaHDAC gene family evolution. The cis-acting element analysis indicated that TaHDACs were involved in hormone response, light response, abiotic stress, growth, and development. Heatmaps analysis of RNA-sequencing data showed that TaHDAC genes were involved in biotic or abiotic stress response. Selected TaHDACs were differentially expressed in diverse tissues or under varying temperature conditions. All selected TaHDACs were significantly upregulated following infection with the barley stripe mosaic virus (BSMV), Chinese wheat mosaic virus (CWMV), and wheat yellow mosaic virus (WYMV), suggesting their involvement in response to viral infections. Furthermore, TaSRT1-silenced contributed to increasing wheat resistance against CWMV infection. In summary, these findings could help deepen the understanding of the structure and characteristics of the HDAC gene family in wheat and lay the foundation for exploring the function of TaHDACs in plants resistant to viral infections.Histone acetylation is a dynamic modification process co-regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Although HDACs play vital roles in abiotic or biotic stress responses, their members in  Histone N-terminal tails harbor a variety of posttranslational modification sites for acetylation, ubiquitination, sumoylation, methylation, phosphorylation, glycosylation, biotinylation, carbonylation, and ADP-ribosylation ,2,3. HisHDAC1, was isolated and cloned from human Jurkat T cells in 1996 [Arabidopsis, rice, maize, barley, and other plants have been identified and characterized [Arabidopsis, 12 of which belong to the RPD3/HDA1-like family, including six members of Class I , five members of Class II , and one member of Class IV (HDA2); two of which belong to SIR2-like family ; and four of which belong to plant-specific HD2-type HDACs  [Triticum aestivum L.) is one of the most important crops worldwide [, and expression of the members of the HDAC gene family in wheat remain unknown.In particular, HDACs are key enzymes involved in the acetylation process and are widely distributed in eukaryotes, including yeasts, animals, and plants. The first histone deacetylation gene, now called human  in 1996 . Since t in 1996 . In the cterized ,13,14. Aorldwide , as it hArabidopsis hda19-1 mutant plants exhibited a disorderly growth [HDACs in maize was highly induced under cold stress, causing the complete deacetylation of the H3 and H4 histones [ZmDREB1 and ZmCOR413 maize cold-responsive genes [Arabidopsis hda6 mutants was found to be significantly lower than that of wild-type plants [HDAC genes were differentially expressed after salt and drought treatment in rice [HDACs might play essential roles in Arabidopsis, maize, and rice in response to abiotic stress, especially low-temperature stress. Therefore, it is particularly important to analyze the relationship between HDACs and the temperature response in wheat.HDACs are reportedly involved in the abiotic stress response ,18. For histones . Maize tve genes . After ce plants . In addi in rice . These fAtHDA19 was induced in Arabidopsis by pathogen-related hormones  and the fungal pathogen Alternaria brassicicola. Overexpression of AtHDA19 increased the expression of the ethylene response factor 1 (ERF1), a key factor in defense response, and enhanced the resistance of transgenic plants against A. brassicicola [AtHDA19 resulted in enhanced plant resistance against Pseudomonas syringae [AtHDA19 mutant was characterized by salicylic acid (SA) accumulation and PR1 and PR2 upregulation, which enhanced the plant tolerance to P. syringae. In contrast, the AtSRT2 gene reportedly negatively regulates plant resistance against P. syringae by inhibiting SA biosynthesis [HDAC family-related genes after the viral infection of wheat would have a certain guiding significance for pathogen-free production of wheat.Besides, HDACs are also involved in response to biotic stress in plants. For instance, the transcription of sicicola . Besidessyringae . The AtHynthesis . In geneynthesis . AlthougHDAC genes in the wheat genome, and analyzed their characteristics, evolutionary relationships, chromosomal location and synteny relationship, protein structure, cis-acting elements, tissue-specific expression levels, response to abiotic or biotic stresses, and expression patterns under temperature variations and viral infection. Silencing TaSRT1 could improve wheat resistance against Chinese wheat mosaic virus (CWMV). This study provided valuable information for the functional investigation of the gene family of T. aestivum HDACs (TaHDAC) and helped us screen candidate genes involved in plant resistance against viral infections.In this study, we identified 49 TaHDAC genes in wheat, according to the AtHDAC gene IDs reported in a previous review [AtHDACs in Arabidopsis thaliana were downloaded from the Arabidopsis Information Resource (https://www.arabidopsis.org). Subsequently, these sequences were used as queries to perform BLASTp and tBLASTn searches  against the wheat reference sequence in the Ensembl Plants database supported by the International Wheat Genome Sequencing Consortium . Next, the coding sequence (CDS) length, the number of exons, and chromosomal locations were determined using Ensembl Plants (http://plants.ensembl.org/tools.html). The molecular weight and isoelectric point (pI) of proteins were predicted using ExPASy (https://web.expasy.org/protparam/), and their subcellular location was predicted using the Bologna Unified Subcellular Component Annotator (BUSCA) webserver (http://busca.biocomp.unibo.it/) [To recognize s review , the amiibo.it/) .AtHDACs and TaHDACs or TaHDACs alone were performed using DNAMAN 6.0 or ClustalW in MEGA7.0 [After sequence screening, multiple sequence alignment of the amino acid sequences of  MEGA7.0 ,29 with TaHDAC genes in wheat chromosomes and gene duplication events, the reference information of the wheat genome was downloaded from NCBI (https://www.ncbi.nlm.nih.gov/genome/?term=wheat) and Ensembl Plants database (http://plants.ensembl.org/Triticum_aestivum/Info/Index). Subsequently, the chromosomal location and synteny relationship was identified using TBtools [To analyze the distribution of  TBtools .https://swissmodel.expasy.org/) [To predict the spatial protein structure, the homology modeling of TaHDAC proteins was performed using the automated SWISS-MODEL homology modeling server (sy.org/) .TaHDAC gene were used to identify their cis-acting elements using the PlantCARE online tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The clustering and arranging of all cis-acting elements were realized with the TBtools [In the putative promoter regions, the 2000 bp sequences upstream of each  TBtools .http://www.wheat-expression.com/) [The expression profile datasets of wheat variety “Chinese Spring” were obtained by the Wheat Expression Browser database (on.com/) ,33. Accoon.com/) .(Triticum aestivum L. cv. Yangmai 158) seeds were soaked in distilled water in a glasshouse at 23 °C with a 16 h light/8 h dark photoperiod. After 1 week(wk), wheat seedlings were transplanted into the soil in small black square pots and used for the viral inoculation assay. When wheat seedlings reached the 3-leaf stage, we applied abiotic stress treatment. Then, we selected wheat seedlings of similar size and placed them in climate chambers at 8 °C, 15 °C, 20 °C, or 25 °C, and tested the expression levels of related genes at 10 days.Wheat 2, 200 mM acetosyringone), the A. tumefaciens was infiltrated into the leaves of N. benthamiana. After 7 days post-inoculation (dpi), the infiltrated leaves were ground in phosphate buffer saline (PBS) for rub-inoculating the 2-leaf stages wheat leaves. The successfully silenced plants were used to inoculate with the CWMV.Foxtail mosaic virus (FoMV)-mediated virus-induced gene silencing (VIGS) had been successfully used in barley and wheat with Nicotiana benthamiana as intermediate host . The spebarley stripe mosaic virus (BSMV) RNAα, β and γ plasmid transcripts were transcribed in vitro and then mixed in equal amounts at a molar concentration ratio of 1:1:1, with excess inoculation buffer (named FES)  as inoculation buffer, as previously described [The linearized escribed . Subsequwheat yellow mosaic virus (WYMV) RNA1 and RNA2 were transcribed in vitro as previously described [The linearized plasmids of CWMV or escribed . Subsequ−ΔΔC(t) method [Triticum aestivum cell division cycle (CDC) gene was used as an internal reference gene [Total RNA was extracted from each wheat sample using a Hipure Plant RNA Mini Kit , according to the manufacturer’s instructions, and stored at −80 °C until use. The first-strand cDNA was synthesized from 1 μg total RNA per 20 μL reaction volume using a First Strand cDNA Synthesis Kit , according to the manufacturer’s instructions, and as previously described . The rea) method . In eachnce gene . All priAtHDAC gene as a query sequence to search for the TaHDAC genes in the wheat genome database. Following the genome-wide search of HDACs, we identified a total of 49 full-length HDAC homologs in wheat. Detailed information about TaHDACs, such as gene ID, location, physical and chemical properties, were listed in TaHDAC genes were located in the nucleus, cytoplasm, chloroplast, mitochondria, and extracellular space. Previous research has shown that the AtHDAC gene family could be divided into 3 categories in Arabidopsis, including the RPD3/HDA1-like, SIR2-like, and HD2 families [AtHDACs and TaHDACs, we created an evolutionary tree and divided them into 5 clades   . Among tionships .TaHDAC genes were unevenly distributed on the 21 chromosomes . Interestingly, TraesCSU02G136000.1 was identified in unknown wheat chromosome (ChrUn). Chr5 contains the largest number of TaHDAC genes (10), whereas Chr4 contains the fewest number of TaHDAC genes (3). 9 TaHDAC genes were detected on Chr2. 7 TaHDAC genes were found on Chr1 and Chr3, respectively. Six TaHDAC genes were found on Chr6 and Chr7, respectively  TraesCS3B02G450300.1/450400.1, TraesCS5A02G114700.3/119300.2, TraesCS5D02G124700.1/126600.1, TraesCS6A02G181100.1/184100.2, TraesCS6B02G210200.1/212600.3, TraesCS6D02G168400.1/171000.1, TraesCS7A02G362600.1/365600.3, TraesCS7B02G261800.1/266000.1, TraesCS7D02G356800.1/360500.1) were clustered into ten tandem duplication event regions on Chr3B/3D/5A/5D/6A/6B/6D/7A/7B/7D  . Our results are illustrated in TaHDAC genes could be divided into 7 categories  and SA [TaHDACs have an analogous function, we used PlantCARE to conduct promoter analysis and identified some cis-acting elements that respond to auxin (IAA), gibberellin (GA), methyl jasmonate (MeJA), ABA, and SA. We noted that the IAA response element mainly included the TGA-element and AuxRR-core, the GA responsive element contained a TATC-box, P-box, and GARE-motif, the MeJA response element covered a CGTCA-motif and TGACG-motif, the ABA response element consisted of ABRE, and the SA response element incorporated a TCA-element and SARE. Among the predicted abiotic stress response elements, the anaerobic induction-related components identified were ARE and GC-motif, LTR, and MBS elements participating in low-temperature response and drought induction, respectively. In addition, we also predicted TC-rich repeat elements involved in defense and stress responsiveness. Finally, an evolutionary analysis revealed that these response elements were unevenly distributed in each clade.The cis-acting elements predicted in the promoter regions of the 49 tegories . Followi) and SA ,39. To iTaHDACs in wheat, we randomly selected a gene from each clade (C I–C V) to analyze their respective expression level in the root, stem, first, second, and third leaves using RT-qPCR analysis (TaHDA6 (TraesCS6A02G181100.1) in C I was found to be downregulated in each tissue. In contrast, the expression level of TaHDA15 (TraesCS5D02G076100.1) in C II was higher, except for its expression level in the stem. The expression levels of TaSRT1 (TraesCS2D02G075800.1) and TaHD2D (TraesCS3B02G450300.1) in C IV and V were shown to be upregulated in various tissues. The expression level of TaHDA2 (TraesCS7B02G266000.1) in C III was observed to be upregulated in the first leaf, whereas its expression level in the second leaf showed no significant change and was downregulated in the stem and the third leaf.To explore the biological functions of analysis . All selTo further clarify the underlying functions of TaHDAC genes response to stresses, the expression profiles were analyzed under heat, PEG6000, Fusarium graminearum, powdery mildew E09, or stripe rust CYR31 treatments using RNA-seq database (Wheat Expression Browser database). After calculation and analysis based on the FPKM values of TaHDAC genes under different stresses and drew the heatmaps . Our resTaHDAC genes might be involved in temperature response. To better understand the mRNA expression level of TaHDACs in wheat under different temperature conditions, we isolated the total RNA from wheat plants grown in a climate chamber  at 10 d, and determined the relative expression of representative TaHDACs using RT-qPCR analysis (TaHDA15 under 20 or 15 °C was not significant change compared to the control (25 °C). However, we could observe several exceptions, such as the expression of TaHDA6/2 at 15 °C were significantly upregulated, whereas them expression level were decreased at 8 °C. Interestingly, the expression of TaSRT1 was shown to be the highest at 20 °C. Likewise, under this temperature condition, the expression of TaHD2D was also increased as expected.Based on cis-acting elements and expression profiles analysis, we found that the analysis . We founTaHDAC genes participate in the stress response to pathogens, it is unknown whether TaHDACs respond to viral infection. To investigate whether TaHDACs respond to plant viral infection, the second leaf (bottom-up) was collected and used in RT-qPCR analysis to study the relative expression patterns of TaHDACs in wheat plants following infection by BSMV, CWMV, and WYMV (TaHDACs tested was shown to be increased relative to the mock (FES buffer), especially that of TaHDA6/2/2D. Strikingly, TaHDA2 expression was significantly increased after BSMV-infection at 13 and 16 dpi . Furthermore, the expression levels of TaHDACs tested after infection of wheat plants with CWMV or WYMV showed an upward trend from 7 to 16 dpi. Subsequently, we also noticed the strongly induced expression (greater than 6.3-fold) of TaSRT1 after CWMV infection at 16 dpi.Although RNA-seq datasets have shown that and WYMV . AccordiAfter analyzing the changes of these selected genes under viral infection, we found that the expression of TaSRT1 gradually increased following CWMV infection and reached the highest expression level at 16 dpi, suggesting that it might play a significant role in the process of CWMV infection. To investigate the role of TaSRT1 in CWMV resistance, we used FoMV-based VIGS technology to silence TaSRT1 in wheat in order to verify its biological function. We inoculated 6 two-leaf stage wheat seedlings with saps of N. benthamiana leaves agroinfiltrated with FoMV + CWMV or FoMV:TaSRT1 + CWMV, respectively. All wheat plants infected with FoMV + CWMV or FoMV:TaSRT1 + CWMV usually showed mosaic symptoms in newly formed leaves, and compared to the FoMV + CWMV plants, TaSRT1-silenced plants exhibited milder symptoms A. FurtheHDAC gene family has been identified and characterized in most organisms, including archaebacteria, eubacteria, fungi, and animals  [Arabidopsis, maize, rice, barley [HDAC gene family the whole-genome level. This study systematically identified 49 HDACs within the Triticum aestivum genome and performed genome-wide identification, phylogenetic analysis, determination of chromosomal locations, and synteny relationships’ protein spatial structures, cis-acting elements, and expression patterns under diverse stress treatments. The AtHDAC gene family in Arabidopsis, includes 3 subfamilies: the RPD3/HDA1-like, SIR2-like, and HD2 families [TaHDACs in wheat. Based on sequence similarity, we further divided the RPD3/HDA1-like family into 3 clades. In general, using phylogenetic analysis in Arabidopsis, these TaHDACs could be divided into 5 clades   ,41,42, a humans) , Arabido, barley , potato , barley , grape [, barley , and tob, barley . However, barley ,48,49 hafamilies ,26, cons5 clades . Our res clades) ; these rlication ,51,52. Aromosome , which l plants. .Arabidopsis HDAC genes were detected to be expressed in 79 different tissues [HDACs were demonstrated to be highly expressive in inflorescences and young floral tissues, but their expression in vegetative tissues was found to be low. The expression profiles of RPD3-like and HD2 subfamilies of HDAC genes were shown to be similar. However, the expression patterns of AtSRT1 and AtSRT2 markedly differed [HDAC genes in rice were shown to be differentially expressed in various tissues. For example, OsHDA703 was mainly expressed in calli and seeds, whereas OsHDA710 was specifically expressed in seedlings and stamens. Besides, both OsHDA706 and OsHDA714 were highly expressed in shoots and leaves [ZmHDA101 was shown to be expressed throughout the germination process, whereas ZmHDA108 was expressed when the cell cycle entered the S-phase [HDACs might be involved in different cell dynamic processes, playing vital roles in various plant species. In our study, all TaHDAC genes tested were found to be differentially expressed in each tissue  were highly expressed in leaf tissues. Moreover, the expression of TaHDA6 and TaHDA2 was demonstrated to be suppressed in the second and third leaves, and stem. In summary, these results implied that TaHDACs might play disparate roles in the growth and development of wheat seedlings.The HDAC proteins are widely expressed in almost all types of plant tissue, such as vegetative tissue, callus, seeds, flowers, and roots. The available microarray analysis data indicated that 16  tissues . Both HDdiffered . In contd leaves . In maiz S-phase . These sh tissue . The expHDACs are involved in the response of plants to abiotic stress [HDAC genes in rice was shown to be regulated by stress-related hormones such as SA, JA, or ABA [HDA6 and HDA19 in Arabidopsis was also found to be induced by JA [HD2A, HD2B, HD2C, and HD2D was shown to be inhibited by ABA and NaCl [Arabidopsis HDA19 has been associated with the ERF3/4/7 transcription repressors in regulating gene expression in response to abiotic stress [axe1-5 AtHDA6 mutant and AtHDA6-RNAi plants, the expression of JA response genes  was reported to be repressed, suggesting that AtHDA6 was a candidate factor for JA response genes [HDAC gene family in wheat contained plant hormones and temperature response elements. Thus, to better understand the gene expression patterns, we also analyzed the cis-acting elements of TaHDAC promoter regions. Our results showed that the TaHDAC promoter regions not only contained phytohormones  but also response elements to abiotic stresses, especially temperature response elements , and compared with the wild type, the hda6 mutant axe1-5 was susceptible to freezing temperatures (−18 °C) after cold acclimation [HDACs in maize was induced by cold stress, resulting in the overall deacetylation of H3 and H4 histones [TaHDA6/2 were upregulated at 15 °C compared with control (25 °C); in particular, the TaSRT1 was significantly upregulated under treatment at 20 °C, suggesting that this gene have a strong response under 20 °C treatment. Moreover, the expression of TaHDA15 at 20 or 15 °C did not exhibit any significant changes, indicating that it was not sensitive to the 2 temperature conditions  and induced the expression of PR1 [TaSRT1-silenced wheat plants inhibited CWMV infection. Therefore, these results further indicated that the HDAC members in different subfamilies of T. aestivum might possess reverse functions under pathogen infection.In addition, studies have reported that esponses ,59. For athogens . The Arapathogen  and the pathogen . Howeverncreased . Altogety mildew ,62. Howen of PR1 . ConsistTaHDACs at the genome-wide level, which could be divided into 5 clades. After analysis of chromosome localization and synteny relationship, we found that TaHDACs were unevenly distributed in the wheat genome, with 21 TaHDAC genes being tandem duplication events, which play a primary role in the expansion of the T. aestivum HDAC gene family. All TaHDACs belonging to the same subfamily had similar protein spatial structures. Results of analysis of cis-acting elements indicated that TaHDACs were involved in hormone response, light response, abiotic stress, and growth and development processes. Analysis of TaHDAC gene expression profiles showed that they were involved in heat, PEG6000, Fusarium graminearum, powdery mildew and stripe rust responses. The expression patterns of the selected TaHDAC genes were shown to be differentially expressed in diverse wheat tissues or under different temperature conditions, and all tested TaHDACs were found to be upregulated following infection with BSMV, CWMV, and WYMV. In addition, the silencing of TaSRT1 enhanced the resistance of wheat against CWMV. These results revealed the HDAC gene family members in wheat and indicated their potential functions in plant resistance to viral infections.In this study, we identified 49


C9orf72-related ALS phenotype appears to be dependent on several modifiers, including demographic factors. Sex has been reported as an independent factor influencing ALS development, with men found to be more susceptible than women. Exposure to both female and male sex hormones have been shown to influence disease risk or progression. Moreover, interplay between genetics and sex has been widely investigated in ALS preclinical models and in large populations of ALS patients carrying C9orf72 repeat expansion. In light of the current need for reclassifying ALS patients into pathologically homogenous subgroups potentially responsive to targeted personalized therapies, we aimed to review the recent literature on the role of genetics and sex as both independent and synergic factors, in the pathophysiology, clinical presentation, and prognosis of ALS. Sex-dependent outcomes may lead to optimizing clinical trials for developing patient-specific therapies for ALS.Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no known cure. Approximately 90% of ALS cases are sporadic, although multiple genetic risk factors have been recently revealed also in sporadic ALS (SALS). The pathological expansion of a hexanucleotide repeat in  Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig’s disease, is a fatal neurodegenerative disease characterized by varying phenotypic manifestations due to degeneration of both upper and lower motor neurons . ClinicaAlthough our understanding of ALS is improving, the pathological pathways that lead to the disease and affect the overall integrity of brain networks are still incompletely understood. Extensive literature provides convincing evidence for a number of cellular and molecular processes found to be implicated in ALS pathogenesis, including, among the most largely studied, excitotoxicity, oxidative stress, impaired protein degradation (proteasome and/or autophagy), toxic protein aggregation, mitochondrial dysfunction, axonal transport, prion-like spreading, dysfunction of non-neuronal cells, neuroinflammation, defects in RNA metabolism, and RNA toxicity . HoweverTwo twin studies, one population- and one clinic-based, have suggested that heritability contributes about 60% to the risk of developing ALS and the unshared environmental component approximately 40% . More resuperoxide dismutase-1 (SOD1) mutations and chromosome 9 open reading frame 72 (C9orf72) repeat expansion has also been explored [Among demographic factors, sex has been reported as an independent factor influencing the development of ALS, with men reported to be 2-3 times more susceptible than women , especiaexplored ,7,8,9,10With this background, in the light of the current need to reclassify ALS patients into pathologically homogenous subgroups potentially responsive to targeted personalized therapies, we aimed to review the recent literature on the role of genetics and sex, as both independent and synergic factors, on the pathophysiology, clinical presentation, and prognosis of ALS. With regard to the search strategy and the selection criteria adopted, we searched Pubmed (2010 to April 2020) using the search terms “amyotrophic lateral sclerosis” or “motor neuron disease” in combination with “epidemiology”, “genetics”, “sex”, and “gender”. Further articles were included from reference lists and review articles. The final reference list was generated on the basis of originality and relevance to the topics covered in this manuscript. Emphasis was placed on publications from the last 5 years but did not exclude highly regarded older publications. C9orf72 (40%), mutations of SOD1 (20%), fused in sarcoma (FUS) (1–5%), and transactive response DNA binding protein (TARBDP) (1–5%), can be most commonly identified [www.ProjectMine.com) [C9orf72 repeat expansion [C9orf72 repeat expansions [An increasing number of genetic variants are recognized as associated with ALS ,11,12. Ientified ,11. In rentified ,14, derientified ,16,17 anine.com) , shed moine.com) ,20, suggine.com) . Moreoveine.com) . Anotherine.com) , and shoxpansion . Of notepansions  or otherpansions ,26.C9orf72 is the most common genetic mutation identified in European and Caucasian patients with ALS, reported in about 40% of patients with FALS, 25% of patients with familial FTD and 5–10% of patients with SALS [C9orf72 expansion influences clinical presentation, with patients carrying the expansion exhibiting higher prevalence of bulbar onset, earlier age at onset, and reduced survival, with higher incidence of comorbid FTD and/or family history of dementia or ALS [C9orf72 has been also associated with Parkinsonism, Huntington phenocopies, Alzheimer’s disease, corticobasal degeneration, and psychosis [C9orf72 is still unknown, there are several theories regarding how the C9orf72 repeat expansion leads to ALS and FTD [transmembrane protein 106B -TMEM106b, ataxin-2- ATXN2, C6orf10/LOC101929163/HLA-DRB1 pathway, and others) [The pathological expansion of a hexanucleotide repeat in ith SALS ,17. C9ora or ALS ,31,32,33sychosis . Althoug and FTD    and FTD ,36; iii) and FTD . Disease and FTD  and the  others) ,39,40.copper/zinc SOD1 gene were identified as causative of ALS in 1993 [C9orf72 repeat expansion account for 0–1.5% of SALS and 5.98% of FALS, SOD1 gene has been revealed as the most common causative gene, accounting for 1.45% of SALS and 25.33% of FALS [SOD1  to date. The reported heterogeneity in the age of onset and phenotype in SOD1-mutated patients [SOD1-mutated patients and in a WES analysis on SALS patients, Pang et al. [SOD1-mutated patients with additional rare variants had shorter survival and that the cumulative effect of rare genetic variants was significant on reducing survival probability in both FALS and SALS.Mutations in  in 1993  and acco in 1993 . Among A of FALS . Current of FALS , and morpatients  and may patients . With reg et al.  observedTARDBP gene, encoding the 43 kDa transactive response DNA-binding protein (TDP-43), are described in 1–5% of FALS [FUS, encoding a second RNA/DNA-binding protein, have been associated with 1–5% of FALS. Similarly to the mechanisms associated with pTDP-43 pathology, FUS toxicity in ALS has been related to dysfunction in RNA processing and in maintaining the DNA damage response [Mutations in the  of FALS . Interes of FALS ,51. pTDP of FALS  and gain of FALS  patholog of FALS . Mutatioresponse ,55.valosin-containing protein (VCP), sequestosome 1 (SQTSM1), ubiquilin 2 (UBQLN2), TANK-binding kinase 1 (TBK1), optineurin (OPTN), coiled-coil-helix-coiled-coil-helix domain-containing 10 (CHCHD10), tubulin alpha 4A (TUBA4A), matrin 3 (MATR3), cyclin F (CCNF), never in mitosis, gene A -related kinase 1 (NEK1), chromosome 21 open reading frame 2 (C21orf2), Annexin A11 (ANXA11), T cell-restricted intracellular antigen-1 (TIA1), and glycosyltransferase 8 domain containing 1 (GLT8D1), has contributed to shedding more light on the genetic architecture of ALS and FTD and the related molecular pathways [TBK1 loss-of-function mutations, producing premature termination codons that cause nonsense-mediated mRNA decay and resulting in the loss of mutant transcript and subsequent loss or reduction of TBK1 protein, has been associated with both ALS and FTD [TBK1 mutated carriers may present ALS and/or FTD with a relatively late age at onset or extrapyramidal symptoms, and associated memory deficits [C9orf72 and GRN mutations, revealed a heterozygous missense mutation in TBK1 in three patients and a loss-of-function mutation in TBK1 associated with deletion of exons 13–15 of OPTN [TBK1 and OPTN are involved in the autophagy pathways) [NEK1 were identified in 3–5% of ALS populations [NEK1 interacts with the chromosome 21 open reading frame 2 (C21orf2) in DNA damage repair, together with vesicle-associated membrane-protein-associated protein B/C (VAPB) and alsin (ALS2) [C21orf2 was also recently identified as an ALS-associated gene [NEK1 and C21orf2 are linked to a skeletal disorder, axial spondylometaphyseal dysplasia [NEK1 carriers, as identified in two Belgian siblings with FALS and cognitive impairment in which NEK1 p.Ser1036* loss-of-function mutation was associated with C9orf72 repeat expansion and TUBA4A p.Thr381Met variant, presenting with early disease onsets [GLT8D1, encoding a widely expressed glycosyltransferase enzyme, has been associated with FALS [More recently, identification of several other genes carrying rare causal variants, including pathways ,56,57. I and FTD ,59. Fromdeficits . Of note of OPTN , therebyathways) . Rare vaulations ,62,63,64ulations  togetherulations  and profulations . Moreoven (ALS2) . Remarkated gene , and autysplasia ,70, furte onsets . More reSOD1, intermediate for C9orf72, and higher for TARDBP) [With this multifaceted genetic background and bearing in mind also the recently proposed “multistep” model of disease in ALS consistent with a six-step process, in the patients carrying genetic mutations associated with ALS, the number of steps necessary to start the neurodegenerative process has been shown to be reduced compared to cases without mutation  support the protective role of progesterone and 17β-estradiol, mainly attributed to their direct effect on neural and muscular cells, preventing cell death, and to a reduction of neuroinflammation. Conversely, androgens seem to affect motoneurons organization, and prenatal exposure to testosterone, irrespective of gender, might play a role in the development of ALS later in life. Finally, neurons spared by ALS  do not show androgen receptors.Findings contrasting with the neuroprotective role of the female hormones ,73,78,88The crude incidence rate of ALS increased by 9% during the 1995 to 2014 period, after age and sex adjustment and mostly limited to women. This increase can be partly ascribed to the effect of the modified lifestyle in women across time, which may have caused a larger exposure of women to exogenous risk factors. No association is described between postmenopausal hormone use and the development of ALS.With regard to cognitive phenotypes, among ALS–FTD patients, women may become more affected than men at increasing age. Moreover, ALS females may exhibit a greater executive impairment than males.SOD1 mutations seem to have a sex-specific effect on the proliferation and differentiation rate of motoneuron cells, according to the study performed in neural progenitor cells (rNPCs) from SOD1 G93A transgenic rats [SOD1 decreased significantly their proliferative and differentiating potential, and sensitivity to oxidative stress was increased, thereby suggesting that sexual dimorphism in ALS can be attributed to intrinsic cellular differences of sex and SOD1 G93A transgene, more than to gonadal steroids. Accordingly, Hayes-Punzo et al. [Several studies have addressed the hypothesis that sex may influence ALS triggers due to genetic mutations in ALS preclinical models and in humans. In particular, nic rats . Li et anic rats  showed to et al.  revealedSOD1 mutations [SOD1 and therefore attenuating microgliosis. Gender differences were hypothesized to be due to a sex-determining region Y element in the CHGB promoter that might act as a suppressor of transcription in males [Another hypothesis regards the role of allelic variants of Chromogranin B (CHGB), a component of secretory vesicles that acts as a chaperone-like protein, in exacerbating sexual differences in the presence of utations . Ohta etutations  found thin males . Of notein males . Moreovein males . In ALS-in males . This moin males . In contin males  reportedin males .mt), induced by misfolded proteins in the IMS and mediated by the estrogen receptor alpha (ERα), was found to upregulate the proteasome and the IMS serine protease Omi  activity [mt [mt [Sex differences in disease phenotype have also been explored in light of the effects on mitochondrial function of mutant SOD1 in male and female ALS transgenic mice and in cellular models overexpressing SOD1 with the G93A mutation . In partactivity . A highevity [mt . Further [mt [mt .More recently, studies on animal models have also been helpful in exploring, together with the motor phenotype, behavior differences between the two sexes: SOD1 G93A males have been shown to display reduced locomotion and exploration and increased anxiety-like behaviors, as well as an increased conditioned cue-freezing response, while impaired intermediate-term spatial memory may be reported in SOD1 G93A females .Crucially, taking into account the sexual dimorphism hypothesized to be involved in pathogenic mechanisms underlying ALS, gender has also emerged as a possible factor influencing response to treatment. As an example, Torres et al.  studied C9orf72 repeat expansion. Remarkably, Rooney et al. [C9orf72 repeat expansion in 4925 ALS cases from Dutch, Irish, and Italian population-based national registers and from two (Belgian and UK) clinical research center cohorts. In this study, the authors revealed a reduced survival rate in male spinal onset patients carrying C9orf72 repeat expansion when compared to females with the same type of onset. Moreover, this group of patients showed a shorter diagnostic delay, suggesting a rapidly progressing disease. These results were in line with those from Trojsi et al. [C9orf72 repeat expansion were younger at onset, at first clinical observation, and at diagnosis and exhibited higher odds of bulbar onset, FTD diagnosis, and family history of ALS, FTD, and Alzheimer’s disease. When stratifying the ALS population by sex, only men carrying C9orf72 repeat expansion showed a shorter survival compared to apparently SALS men  carriers, a recent meta-analysis on sex differences in genetic mutations in ALS and FTD showed that in women there has been a higher prevalence of expanded C9orf72-related ALS [C9orf72-related ALS phenotype [C9orf72 expansions has been shown to increase with age [C9orf72 expansion have been recognized to be more prone to develop ALS at a younger age [C9orf72-related ALS may have less exposure to these risk factors [C9orf72 mutations probably may have a shorter disease course [With regard to evidence of potential sex–genetics interplay in ated ALS . This fihenotype ,104, conwith age ,27. Convnger age . Currentnger age ,107, ande course .C9orf72 repeat expansion. In a population-based study by Chiò et al. [C9orf72 repeat expansion carriers.Other studies did not confirm the potential interaction between sex and occurrence of ò et al. , geneticò et al.  did not ALS is a complex disease, and this complexity is mostly due to the heterogeneity of its phenotype, influenced by genetics through oligogenic mechanisms, that is, through gene–gene (or protein–protein) interactions ,19 and bC9orf72-related ALS, gain of neurotoxic protein (RNA or dipeptide repeat) aggregates. Moreover, longitudinal incidence studies could be a relevant manner through which sex differences in survival and disease course may be accounted for when examining genetically well-characterized ALS populations, especially for C9orf72-related ALS.Most differences between the two sexes in the pathogenic mechanisms triggering ALS have been reported in preclinical models, mainly investigating disease pathways in SOD1G93A transgenic mice. Future research should explore potential sex differences in the ALS-related pathogenic mechanisms, such as, with regard to


Lactobacillus brevis Bmb6 alleviates colitis by improving the tight junction (TJ) of the inflamed intestinal epithelial layer. In the present study, the regulatory effects of L. brevis Bmb6 on the TJ barrier to ameliorate colitis-symptoms were investigated. Preliminary screening showed that L. brevis Bmb6 exhibited strong acid and bile acid tolerance, along with antioxidants and β-galactosidase activities. In a 14-day dextran sulfate sodium (DSS)-induced colitis mouse model, treatment with L. brevis Bmb6 significantly decreased in the disease activity index score. In addition, histological analyses showed that treatment with L. brevis Bmb6 protected the structural integrity of the intestinal epithelial layer and mucin-secreting goblet cells from DSS-induced damage, with only slight infiltration of immune cells. Interestingly, western blotting analyses showed that the expression of the TJ protein, zona occluden-1, was restored in Bmb6-treated mice, but not in DSS-induced mice. Consistently, the gene expression of inflammatory cytokines (tumor necrosis factor-α and interferon-γ) was also suppressed in the Bmb6-treated mice. Hence, our findings suggest that suppression of inflammatory conditions enhanced expression of TJ protein, ZO-1, or vice versa, contributing to a colitis-ameliorating effect in L. brevis Bmb6.The integrity of gut barrier functions is closely associated with the pathogenesis of colitis. It is speculated that  Inflammatory bowel disease (IBD) is a gastrointestinal tract disorder characterized by chronic inflammation of the mucosal cells. The incidence of IBD has been increasing rapidly since 1990, affecting one in 200–300 people in high-income countries. These epidemiological data suggest that a further increase in IBD incidence may pose a major health burden to the community . InteresAs vital components of the epithelial barrier, TJ proteins such as claudins, occludin, and zonula occludens (ZO) connect the adjacent epithelial cells and provide mechanical stability to facilitate intercellular communication and paracellular transport . For insLactobacillus has been used as an indicator of healthy gut and for the treatment for various diseases, especially those related with gastrointestinal discomforts, such as antibiotic-associated dysbiosis, IBD, and lactose intolerance. Numerous studies have reported that treatment with Lactobacillus species ameliorates colitis by regulating host immune responses, primarily via suppressing the overexpression of inflammatory factors  daily. At day 7, the drinking water in the DSS and Bmb6 groups was replaced with 4% (w/v) DSS in water until the end of the experiment. The disease activity index (DAI) was assessed daily based on a scoring system . Eighteen five-week-old female C57BL/6J mice were purchased from Daehan Lab . The mice were housed and acclimatized for one week in the Animal Housing Unit under standard conditions of 22–25 °C, 50–60% humidity, and 12 h light/dark cycle. Standard mouse chow-diet and water were provided ad libitum. The mice were divided into three groups: control, DSS, and Bmb6 groups, with six mice in each group  and stained with hematoxylin-eosin for histological evaluation. For mucosal layer evaluation, Alcian blue was used to stain the mucin in the paraffin-embedded sections and the nuclei were counterstained with nuclear fast red. For immunofluorescence analysis, the cut sections were stained with ZO-1  or claudin-1 antibodies  and goat anti-rabbit Alexa Flour 488 secondary antibody . 4′,6-diamidino-2-phenylindole  was used to counterstain cell nuclei. Slides were examined and analyzed using an epifluorescence microscope.Total RNA was extracted from the excised mouse colon using the RNeasy Mini Kit  according to manufacturers’ protocol. Next, 2 μg of total RNA was used to synthesize complementary DNA (cDNA) using Maxime RT Premix Oligo (dT) RT-PCR kit . The primers used in the study are listed in g at 4 °C for 5 min, and the supernatant was collected as the extracted protein. The concentration of the extracted protein was determined using the Pierce™ BCA Protein Assay Kit . The extracted proteins (50 µg) were separated using 12% sodium dodecyl sulfate-polyacrylamide gel and electroblotted  onto the polyvinylidene difluoride membranes . The membranes were blocked with ZO-1 , claudin-1 , and β-actin  antibodies in 5% (w/v) skim milk-supplemented with Tween 20-Tris-buffered saline (TTBS) overnight at 4 °C. After incubation, membranes were washed three times with TTBS and incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit antibodies. Protein bands were then developed and detected with enhanced chemiluminescence, and the band density was determined using β-actin as the reference protein.Total protein was extracted from the excised mouse colons using PRO-PREP protein extraction solution . Briefly, 100 mg of the excised mouse colon tissue was immersed in 500 mL of PRO-PREP solution and homogenized using Q125 sonicator  for 5 min (pulsing mode of 15 s on and off) and 40% power in the ice bath. The mixture was then centrifuged at 13,000× n = 3 or 6). Tukey’s multiple comparison test and repeat measure analysis of variance (ANOVA) were performed using SPSS 20  with a p < 0.05 considered to be statistically significant.All data are presented as the mean ± standard deviation from three independent runs or six animals per group  ox-gall.As shown in Lactobacillus strain Bmb6 was the most effective DPPH radical scavenger (59.48%), as compared to strains R10 (23.59%) and P11 (29.85%). The SOD assay results  was constructed based on the 16S rDNA, and a close grouping of Bmb6 with the other eighteen L. brevis strains was observed, which was in agreement with the BLAST results, thus validating its identity as L. brevis Bmb6.16S rDNA analysis using BLAST showed that strain Bmb6 had 100% homology with L. brevis Bmb6-treated mice display higher alleviation of intestinal hemorrhage than DSS-induced mice.The control mice exhibited the longest colon length (7.35 ± 0.68 cm), with normal shaped feces in the light red color colon a,b. In cL. brevis Bmb6 significantly alleviated colitis symptoms, as loose and hemoccult feces and a lower DAI score of 4.33 ± 0.58 was observed at the end of the study, as compared to the DSS group. Meanwhile, the control group showed no colitis symptoms, and the DAI score fluctuated around 1.0.On day-10, the DAI score began to rise in the DSS group, and loose, unformed, and bloody stools were visible to the naked eye c. The DAL. brevis Bmb6-treated mice was greatly improved, with only slight epithelial structural damage and immune cell infiltration.Hematoxylin-eosin-stained colon sections showed normal and intact intestinal epithelial structures with no symptoms of immune cell infiltration in the control group . HoweverThe Alcian blue-stained colon micrographs  showed tL. brevis Bmb6 ameliorates colitis by partially recovering the expression of ZO-1 protein.As shown in L. brevis Bmb6 suppressed IFN-γ expression to a level similar to that in control. Moreover, TNF-α gene expression was significantly suppressed in the Bmb6 group compared to that in the DSS group but remained at a higher level than the control group. Meanwhile, no difference in interleukin (IL)-1β gene expression was observed between the control, DSS, and Bmb6 groups.As shown in Bifidobacterium, Lactobacillus, Lactococcus, and Pediococcus, and their bioactive metabolites found in fermented food, have shown to having been responsible for these functions [L. brevis Bmb6 possess several probiotic traits, including tolerance to acidic and bile acid conditions, which resemble the gut environment, and a prominent β-galactosidase activity.Numerous studies have reported that consumption of fermented food helps in alleviating gastrointestinal disorder symptoms ,30,31,32unctions ,34,35. HL. brevis Bmb6 possesses strong antioxidant potential, as indicated by its prominent radical scavenging activity in the preliminary DPPH scavenging and SOD assays. A growing body of evidence suggests that elevated reactive oxygen and nitrogen species are closely associated with intestinal inflammation [Lactobacillus significantly reduces colonic myeloperoxidase level, oxidative stress, and inflammation in DSS-induced colitis mice [L. brevis Bmb6 suggest its potential role in alleviating colitis, its effect on lowering oxidative stress in vivo has not yet been evaluated.In addition, ammation . Accumulammation ,38. Sevetis mice ,40,41,42L. brevis Bmb6-containing fermented milk significantly improved the DAI score in DSS-induced mice [L. brevis Bmb6 alleviated colon hemorrhage while preserving the intact intestinal epithelial structure but showed no effect on restoring the colon length. Histological analyses revealed that L. brevis Bmb6 treatment alleviated colonic hemorrhage in DSS-induced colitis mice by preserving the integrity of the epithelial structure and preventing mass infiltration of immune cells and destructive damage of goblet cells in the colon, thereby contributing to the lower DAI score and absence of bloody diarrhea in the Bmb6 group. Preservation of goblet cells is crucial for maintaining mucosal barrier integrity via the secretion of mucins and trefoil factors [L. brevis Bmb6 was partly attributed to its ability to preserve the integrity of the intestinal epithelial structure.For in vivo evaluation, DSS was used to induce colitis in mice. The clinical symptoms of DSS-induced mice are similar to those of patients with IBD, which include bloody stool, diarrhea, elevated inflammatory biomarkers, and drastic weight loss . In our ced mice . This isced mice ,45,46. I factors . TherefoL. brevis Bmb6 significantly improved the expression of ZO-1 in the Bmb6 group, indicated by western blot analysis. Hence, our findings indicate a strong correlation between ZO-1, DAI score, and colon tissue histology, highlighting the role of L. brevis Bmb6 in TJ recovery and preservation of the intestinal epithelial structure, thereby ameliorating colitis.The expression and localization of TJ proteins, such as ZOs, occludin, and claudins play a key role in regulating intestinal barrier functions. Impairment of the TJ barrier increases the paracellular permeation of pro-inflammatory molecules and activates mucosal immune response, resulting in chronic inflammation and tissue damage ,48,49,50In addition to ZO-1, the expression of claudins, which are critical components of the TJ barrier, was also investigated in the present study. Claudins connect adjacent cells via heterophilic and hemophilic interactions, forming pores and barriers for paracellular permeation of specific molecules in various tissues ,53,54. FκB and initiates the pro-inflammatory cascade by recruiting other pro-inflammatory factors, such as IFN-γ and IL-1β, further intensifying inflammatory reaction and intestinal dysfunction [L. brevis Bmb6 significantly suppressed the gene expression of TNF-α and IFN-γ, but not IL-1β in DSS-induced colitis mice. These outcomes were consistent with the histological analyses data showing improved protein expression of the TJ protein, ZO-1, and well-preserved intestinal epithelial structure in L. brevis Bmb6-treated DSS-induced colitis mice, thereby indicating that regulation of inflammation conditions can restore the expression of TJ protein or vice versa.Abnormal and uncontrolled secretion of inflammatory cytokines is also commonly seen in patients with IBD. Among the vast number of cytokines, TNF-α plays a key role in the pathogenesis of IBD by inducing inflammation and cell apoptosis, and subsequently intestinal TJ barrier defect in intestinal epithelial cells ,63. As afunction ,64. Therfunction ,66,67. HL. brevis Bmb6 isolated from the fermented food, kimchi, exerted significant colitis-ameliorating effects through suppression of pro-inflammatory cytokines, improvement of ZO-1 protein expression, and preservation of the intestinal epithelial structural integrity. Our findings showed the colitis-ameliorating effects in L. brevis Bmb6 could be attributed to a close association between inflammation and TJ barrier functions, wherein a reduction in inflammatory response can restore the expression of TJ protein, ZO-1, and vice versa. Notably, the expression of claudin-1 remained controversial in the present experimental colitis model. Further investigation is needed to clarify the role of claudin-1 in IBD pathogenesis.Taken together,


There are no established molecular biomarkers for patients with breast cancer receiving combination endocrine and CDK4/6 inhibitor (CDK4/6i). We aimed to determine whether genomic markers in circulating tumor DNA (ctDNA) can identify patients at higher risk of early progression on fulvestrant therapy with or without palbociclib, a CDK4/6i.PALOMA-3 was a phase III, multicenter, double-blind randomized controlled trial of palbociclib plus fulvestrant (n = 347) vs placebo plus fulvestrant (n = 174) in patients with endocrine-pretreated estrogen receptor–positive (ER+) breast cancer. Pretreatment plasma samples from 459 patients were analyzed for mutations in 17 genes, copy number in 14 genes, and circulating tumor fraction. Progression-free survival (PFS) was compared in patients with circulating tumor fraction above or below a prespecified cutoff of 10% and with or without a specific genomic alteration. All statistical tests were 2-sided.P = .004) and placebo plus fulvestrant . In multivariable analysis, high-circulating tumor fraction was associated with worse PFS , as was TP53 mutation  and FGFR1 amplification . No interaction with treatment randomization was observed.Patients with high ctDNA fraction had worse PFS on both palbociclib plus fulvestrant (hazard ratio [HR] = 1.62, 95% confidence interval [CI] = 1.17 to 2.24; Pretreatment ctDNA identified a group of high-risk patients with poor clinical outcome despite the addition of CDK4/6 inhibition. These patients might benefit from inclusion in future trials of escalating treatment, with therapies that may be active in these genomic contexts. CDK4/6 inhibitors (CDK4/6i) now play a key role in the treatment of advanced, estrogen receptor–positive (ER+) breast cancers , with esHER2 amplification associated with poorer outcome in primary disease include mutations in TP53  is found in the plasma of a substantial majority of patients with advanced cancer and presents a source of cancer DNA for noninvasive analysis of tumor somatic genetic features. In addition, circulating tumor fraction, the fraction of plasma DNA that is derived from the tumor, may be a biological marker that reports on both tumor bulk and tumor aggressiveness  and is aIn conducting this analysis, we hypothesized that genomic aberrations identified at baseline, including mutations, copy number, and circulating tumor fraction, could be predictive or prognostic of clinical outcome for patients with advanced ER+ breast cancer receiving fulvestrant with or without palbociclib. We investigated this using a multimodal ctDNA sequencing analysis of plasma DNA from the PALOMA-3 trial.Full details of the methods can be found in the The design of the PALOMA-3 trial NCT01942135) and clinical outcome data has been previously reported  assay directed at CDK4, CDK6, CDKN1A, CDKN1B, RB1, and NF1; exons 5-8 of TP53, and mutation hotspots in AKT1, ERBB2, ESR1, PIK3CA, FGFR1, FGFR2, FGFR3, KRAS, HRAS, and NRAS. Of the baseline plasma DNA sequencing, 195 patients were previously sequenced to compare mutational profile with end-of-treatment progression plasma  within both treatment arms. PALOMA-3 was designed and powered for a clinical endpoint and, as such, was not specifically powered for a translational analysis. Survival analyses to associate PFS with genomic aberrations were performed with Cox proportional hazards models, with calculation of hazard ratios (HR), 95% confidence intervals (CI), and log-rank  Thernau . For cir Thernau ,24. To er = 0.86; P < .001; PIK3CA allele fraction  patients had sufficient material and subsequent library quality for circulating tumor fraction and copy number analysis, a group with outcomes representative of the overall trial population  was observed in 38.9% (156 of 401) patients . In the ESR1  and TP53 (52 of 331 [15.7%]). Smaller proportions of patients had mutations in NF1 (21 of 331 [6.3%]), ERBB2 (12 of 331 [3.6%]), and AKT1 (10 of 331 [3.0%]). Mutations in ESR1 were polyclonal in a subset of patients  had sufficient material and subsequent library quality for mutation analysis by sequencing, with this population also representative of the overall trial , PTEN (30 of 156 [19.2%]), and CDKN2A (34 of 156 [22.0%]).Detection of copy number aberrations (CNAs) is technically challenging in samples with low circulating tumor fraction, and we assessed the prevalence of CNAs only in the group with more than 10% circulating tumor fraction. The most frequently observed gains from the genes included in the panel were nd FGFR1 , and theESR1 mutation and previous endocrine sensitivity (χ2P = .015), previous aromatase inhibitor exposure (χ2P = .002), bone metastases (χ2P = .005), and a number of all previous lines of treatment for metastatic disease (Cochran-Armitage P = .02), associations similar to those previously reported using digital PCR analysis (2Q = .021) and bone metastases (χ2Q = .028) remained statistically significant after correction for multiple testing using the Benjamini-Hochberg method. TP53 mutations were positively associated with visceral metastases (χ2Q = .046), soft tissue and lymph node metastases (χ2Q = .042), and a number of disease sites (Cochran-Armitage Q = .009). No other mutations or copy number changes were statistically significantly associated with a particular clinical characteristic after correction for multiple testing. There was no detectable association between circulating tumor fraction of more than 10% and clinical and pathological features, after correcting for multiple comparisons . Multiple copy number gains were associated with poorer PFS including MCL1 gain , FGFR1 gain , MYC gain , CDK4 gain , and CCNE1 gain . Loss and/or loss of heterozygosity of RB1, PTEN, and CDKN2A were also associated with worse prognosis  and ESR1 mutations  were associated with worse PFS after correction for multiple testing using the Benjamini-Hochberg method. Copy number gain (amplification) of FGFR1  and MCL1  were associated with worse PFS. With a 2:1 randomization, the placebo plus fulvestrant group was relatively small, making direct comparisons between treatment groups challenging, and there were no individual aberrations that had a statistically significant interaction P value with treatment.In the group of patients treated with placebo plus fulvestrant , along with TP53 mutation  and FGFR1 gain . Patients with TP53 mutations and FGFR1 amplifications had a very poor median PFS on palbociclib and fulvestrant of 3.7 months and 3.9 months, respectively  patients , a group who may benefit from augmented treatment strategies.Combinations of CDK4/6i and endocrine therapy are standard of care in advanced ER+ breast cancer. There are few molecular markers available to identify patients at risk of early progression, where increased monitoring to detect progression may be appropriate and for whom research efforts might be focused to improve outcomes. We have previously published work from the PALOMA-3 trial examining the evolution of resistance . Here, wital PCR . We did PIK3CA and TP53, although the association was weaker at lower mutation allele fractions, likely reflecting subclonal mutations and stochastic effects , Breast Cancer Now with support from the Mary-Jean Mitchell Green Foundation and Pfizer. We acknowledge National Institute for Health Research funding to the Royal Marsden and Institute of Cancer Research Biomedical Research Centre. We thank Breast Cancer Now for funding this work as part of Program Funding to the Breast Cancer Now Toby Robins Research Centre.Role of the funder: The sponsor collaborated with the senior academic authors to design the clinical study and assisted with data collection, analysis, and interpretation. All authors had responsibility for the decision to submit the manuscript for publication.Disclosures: BO’L—Research funding (Inst): Pfizer. YL, XH, CHB—Ex-Employment: Pfizer, Stock ownership: Pfizer. FA—Travel, Accommodation, Expenses: Novartis, Roche, GlaxoSmithKline, AstraZeneca, Research Funding (Inst): AstraZeneca, Novartis, Pfizer, Lilly, Roche. SLoib—Consulting or Advisory Role (Inst): Pfizer, Roche, Novartis, Seattle Genetics, Honoraria: Pfizer, Roche, Research Funding (Inst): Pfizer, Roche, Celgene, Amgene, Novartis, Abbvie, AstraZeneca, Seattle Genetics, Teva, Vifor Pharma. SLoi—Consulting or Advisory Role (Inst): AstraZeneca/MedImmune, Seattle Genetics, Bristol-Myers Squibb, Pfizer, Novartis, Roche/Genentech, Merck Sharp & Dohme, Research Funding (Inst): Roche/Genentech, Pfizer, Novartis, Merck, Puma Biotechnology, Bristol-Myers Squib. MC—Consulting or Advisory Role: Dompé Farmaceutici, Newomics, Vortex Biosciences, Honoraria: Dompé Farmaceutici, Pfizer. NC—Consulting or Advisory Role: Roche, Pfizer, Novartis, AstraZeneca Research Funding: Pfizer (Inst), Roche (Inst), AstraZeneca. The other authors have no conflicts of interest to discloses.Author contributions: BO’L: conceived and designed the project; collected and assembled the data; data analysis and interpretation; writing manuscript. RJC: collected and assembled the data; data analysis and interpretation; writing manuscript. XH: data analysis and interpretation; writing manuscript. SH: collected and assembled the data; data analysis and interpretation; writing manuscript. YL: conceived and designed the project; collected and assembled the data; data analysis and interpretation; writing manuscript. FA: collected and assembled the data; writing manuscript. SLoib: collected and assembled the data; data analysis and interpretation; writing manuscript. SLoi: collected and assembled the data; data analysis and interpretation; writing manuscript. IG-M: conceived and designed the project; collected and assembled the data; data analysis and interpretation; writing manuscript. MC: conceived and designed the PALOMA-3 study; collected and assembled the data; data analysis and interpretation; writing manuscript. CHB: conceived and designed the PALOMA-3 study; conceived and designed the project; collected and assembled the data; data analysis and interpretation; writing manuscript. NCT: conceived and designed the PALOMA-3 study; conceived and designed the project; collected and assembled the data; data analysis and interpretation; writing manuscript.Acknowledgments: We would like to thank the patients, families, and care givers who participated in the PALOMA-3 trial and all the investigators and site personnel.The data that support the findings of this study are available from the corresponding author on reasonable request.djaa087_Supplementary_DataClick here for additional data file.


The bipolar mitotic spindle drives accurate chromosome segregation by capturing the kinetochore and pulling each set of sister chromatids to the opposite poles. In this review, we describe recent findings on the multiple pathways leading to bipolar spindle formation in fission yeast and discuss these results from a broader perspective. The roles of three mitotic kinesins  in spindle assembly are depicted, and how a group of microtubule-associated proteins, sister chromatid cohesion and the kinetochore collaborate with these motors is shown. We have paid special attention to the molecular pathways that render otherwise essential Kinesin-5 to become non-essential: how cells build bipolar mitotic spindles without the need for Kinesin-5 and where the alternate forces come from are considered. We highlight the force balance for bipolar spindle assembly and explain how outward and inward forces are generated by various ways, in which the proper fine-tuning of microtubule dynamics plays a crucial role. Overall, these new pathways have illuminated the remarkable plasticity and adaptability of spindle mechanics. Kinesin molecules are regarded as prospective targets for cancer chemotherapy and many specific inhibitors have been developed. However, several hurdles have arisen against their clinical implementation. This review provides insight into possible strategies to overcome these challenges. The bipolar mitotic spindle is a dynamic ensemble of core microtubule polymers and a cohort of microtubule-associated proteins (MAPs). It attaches to the kinetochore on sister chromatids to align chromosomes at the spindle equator in metaphase, and pulls each pair of sister chromatids towards the opposite poles in anaphase A. During anaphase B, spindles further elongate to ensure the equal partition of each set of chromosomes, which is followed by cytokinesis ,2. MAPs Aspergillus nidulans as one of the mitotically arrested mutants  [Aspergillus BimC, C. elegans BMK-1, Drosophila Klp61F, Xenopus Eg5 and human KIF11) is essential for mitosis, in which any means of its inactivation, e.g., chemical inhibition, genetic deletion or RNAi-mediated depletion, leads to the emergence of monopolar spindles, the failure of chromosome segregation and viability loss [The type 5 kinesin (Kinesin-5) was originally identified in ed bimC) . This kied bimC) ,10. Duried bimC) ,12. In mity loss ,17,18,19Aspergillus nidulans and Saccharomyces cerevisiae showed that lethal mutations in Kinesin-5 are rescued by simultaneous inactivation of genes encoding Kinesin-14 (klpA and KAR3 respectively) [Aspergillus KlpA, Drosophila Ncd, Xenopus XCTK2 and human HSET/KIFC1) or Dynein [Surprising findings came to light that cells can divide in the absence of Kinesin-5 function under certain conditions across a wide range of species, including human beings, frogs, flies, filamentous fungi and the budding and fission yeasts. Initial genetic studies and recent more comprehensive analysis conducted in ctively) ,21,22,23r Dynein . Accordicut7 temperature sensitive (ts) mutants in fission yeast. Spontaneous survivors of cut7 mutant strains grown at the restrictive temperature (36 °C) were isolated. After standard genetic analyses and nucleotide sequencing, suppressor genes (designated skf = suppressor of kinesin five) were identified , atb2 (α2-tubulin) and nda3 (β-tubulin) [alp7 (encoding an orthologue of the transforming acidic coiled-coil protein TACC) [alp14 and dis1 (two genes encoding XMAP215/Stu2/TOG microtubule polymerases) [We have found that mutations in the genes encoding tubulins and five non-motor MAPs are also capable of rescuing tubulin) ,42 (Tabltubulin) . Its muttubulin) ,45. Alp1tubulin) ,47,48. Tin TACC) ,50, alp1merases) ,54,55,56merases) ,54,57. Tmerases) . cut7 ts mutation by the mal3 mutation could be ascribable to the loss of Klp2 function [cut7 mutant cells, the intensities of spindle microtubules are augmented [cut7 ts and any of the suppressor mutations, the intensities of Klp2 are reduced. Given these observations, we propose that the rescue of cut7 mutants by these suppressors is derived from quantitative downregulation of Klp2 activity. In fact, the overproduction of Klp2 in the double mutants between cut7 and suppressor mutations restored a ts phenotype similar to a single cut7 mutant [cut7 mutation. In summary, suppressor analyses have uncovered that multiple factors that regulate microtubule structures are involved in several mechanisms by which Kinesin-5/Cut7 becomes dispensable, and importantly, inactivation of Kinesin-14s, Pkl1 and Klp2, is the main means for the rescue of cut7 by these suppressors. Apart from its role in microtubule stabilisation, Mal3/EB1 is known to interact with Klp2, which is a prerequisite for this motor to be loaded on the spindle microtubule . Thus, sfunction . Overallugmented . Importa7 mutant , corrobocut7 mutants exhibit increased intensities of the spindle microtubule accompanied by more Klp2 proteins on the spindle microtubule. In line with this observation, these cells display hyper-resistance against microtubule-depolymerising drugs, such as thiabendazole (TBZ) or methyl 2-benzimidazolecarbamate (MBC), and interestingly, treatment of cut7 ts mutants with TBZ or MBC rescues temperature sensitivity [cut7. Collectively, the impairment of microtubule stability and/or dynamics by either suppressor mutations or treatment with microtubule-destabilising drugs renders fission yeast cells viable in the absence of Kinesin-5 , microtubule destabilisation can also effectively rescue monopolar spindle phenotypes induced by Kinesin-5/KIF11 inhibition ,60. Thispka1 gene rescues the cut7 ts mutant [cut7 ts mutant as pka1∆ is [cut7 mutation to a bipolar spindle, leading to rescue of this mutant. In many organisms, the extracellular environment, such as nutritional cues, regulates microtubule dynamics through intracellular signal transduction pathways. In yeasts, glucose in the media activates the cAMP/PKA pathway, by which it controls a diverse set of downstream events ,62,63. Ds mutant . Pka1 res mutant ,67,68,69pka1∆ is . Cls1/Pepka1∆ is . It is pcut7∆pkl1∆) or if microtubules are destabilised . This finding poses the following important question: how do bipolar spindles assemble in the absence of Kinesin-5-mediated outward force? Detailed genetic and cell biological analyses have unravelled this puzzle; at least 11 gene products are capable of generating outward forces in place of Kinesin-5  phenotype [One possibility of the survival of otubules ,72. Prevotubules , though otubules ,74,75. Dhenotype . How do the two N-kinesins, Kinesin-5/Cut7 and Kinesin-6/Klp9, act in concert to drive spindle elongation in wild-type cells? Differential localisation patterns of these two kinesins on the spindle microtubule may give us a clue. While Cut7 mainly accumulates near the SPB, Klp9 is localised exclusively to the spindle midzone where antiparallel microtubules interdigitate A. Given Fission yeast cells are capable of forming nearly normal bipolar spindles in the presence of only Kinesin-6 Klp9, which acts in spindle elongation later in mitosis . How theC. elegans does not require Kinesin-5/BMK-1 for bipolar spindle formation [Unlike in other species, ormation . During ormation . Interesormation ,84. Takecut7∆pkl1∆alp14∆, cut7∆pkl1∆dis1∆ or cut7∆pkl1∆alp7∆, are inviable. SPB-localising Csi1 and Csi2 are required for Alp7 localisation to the mitotic SPBs [csi1 or csi2 is lethal in combination with cut7∆pkl1∆. Temperature sensitive cut7∆pkl1∆alp7 cells display monopolar spindles or very short spindles (<0.5 μm) that fail to elongate [cut7∆pkl1∆ cells outward forces are generated through microtubule polymerisation, in which the growing plus ends of the microtubule push the SPB, leading to separation of the SPBs , which ensures the temporal order of bipolar spindle assembly to be synchronised with mitotic onset. Implementation of dual, redundant pathways might be also beneficial for the robustness of the system, disrupting one pathway would not result in a catastrophic impact on spindle formation and therefore the fidelity of chromosome segregation. Xenopus oocytes, where the Ran GTPase acts as a master regulator [Xenopus oocyte and human acentrosomal cells, Kinesin-5 crosslinks the antiparallel microtubules while Kinesin-14 and/or Dynein are required for spindle pole focusing [In higher eukaryotes and plants, the bipolar spindle is formed independent of the centrosome through the pathway referred to as the acentrosomal pathway ,113,114.egulator ,116. Thiegulator ,118,119.focusing , two proKinesin-5 is required for successive cell division. Furthermore, in actively growing cancer cell lines, its activity is tightly, and sometimes causally, linked to tumour progression and malignancy . Accordi