Saussurea costus is a plant traditionally used for the treatment of several ailments. Our study accomplished the UPLC/T-TOF–MS/MS analysis of a methanol extract of Saussurea costus roots (MESC), in addition to lipoidal matter determination and assessment of its in vivo hepatoprotective activity. In this study, we were able to identify the major metabolites in MESC rather than the previously known isolated compounds, improving our knowledge of its chemical constituents. The flavones apigenin, acacetin, baicalein, luteolin, and diosmetin, and the flavonol aglycones quercetin, kaempferol, isorhamnetin, gossypetin, and myricetin and/or their glycosides and glucuronic derivatives were the major identified compounds. The hepatoprotective activity of MESC was evaluated by measuring catalase activity using UV spectrophotometry, inflammatory cytokines and apoptotic markers using ELISA techniques, and genetic markers using PCR. Paracetamol toxicity caused a significant increase in plasma caspase 2, cytokeratin 18 (CK18), liver tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), miRNA-34a, and miRNA-223, as well as a significant decrease in liver catalase (CAT) activity and in the levels of liver nuclear factor 1α (HNF-1α), sirtuin-1, and C/ebpα. Oral pretreatment with MESC (200 mg/kg) showed a significant decrease in caspase 2, CK18, TNF-α, IL-6 and a significant increase in liver CAT activity. MESC decreased the levels of liver miRNA-34a and miRNA-223 and induced HNF-1α, sirtuin-1, and C/ebpα gene expression. The histological examination showed a significant normalization in rats pretreated with MESC. Our findings showed that Saussurea costus may exert a potent hepatoprotective activity through the modulation of the expression of cellular cytokines, miRNA-34a, and miRNA-223. Saussurea costus  is considered one of the most important traditional Chinese medicinal plants. It is a rich source of various bioactive phytoconstituents, and its genus comprises about 300 species +. Luteolin-O-hexoside (21) exhibited a deprotonated molecule [M-H]− at m/z 447.0966; we then observed a subsequent loss of 162 amu for hexose at m/z 285.0520 [M-H-hexose]−, while luteolin-C-hexoside (31) showed a protonated molecule [M+H]+ at m/z 449.1549. Luteolin-di-O-hexoside (13) exhibited a deprotonated molecule [M-H]− at m/z 609.1462; moreover, luteolin aglycone (40) was detected with a deprotonated molecule [M-H]− at m/z 285.039, and characteristic peaks at m/z 269.1614 [M-H-OH]− and 151.0980 indicated a dihydroxy-substituted A-ring [The glycoside diosmin (24) exhibited a protonated molecule [M+H]hexoside  exhibitem/z 577.1506 and a fragment at m/z 269.0386 [M-H-neohesperidose]−. Apigenin-C-hexoside (22) showed a protonated molecule [M+H]+ at m/z 433.1232; however, apigenin-O-hexoside (32) showed a protonated molecule [M+H]+ at m/z 433.1851 and a fragment ion peak at m/z 271.1296 [M+H-hexose]+. Moreover, apigenin aglycone (34) was detected at m/z 269.0434 [M-H]− [O-rutinoside (10) showed a protonated molecule [M+H]+ at m/z 593.1631; on the other hand, acacetin aglycone (39) exhibited a deprotonated molecule [M-H]− at m/z 283.1895 and a production at m/z 253.1469 corresponding to [M-H-OCH3]−. Baicalein-O-glycuronide (14) showed a protonated molecule [M+H]+ at m/z 477.0679.Rhoifolin glycoside (Apigenin neohesperidoside) (20) showed a deprotonated molecule [M-H] at hexoside  showed a+ at m/z 319.1651 [m/z 481.1810 [M+H]+ and also showed a fragment at m/z 319.0663 [M+H-hexose]+. Quercetin hexoside (19) showed a protonated molecule [M+H]+ at m/z 465.1058; The neutral loss of 162 amu of the hexose moiety was observed in MS2 fragmentations at m/z 303.0471 [M+H-hexose]+. Quercetin glucuronide (11) was detected at m/z 477.0659 [M-H]−, and the neutral loss of 176 Daltons of the glycuronic moiety was confirmed by an ion at m/z 301.0332 [M-H-glycuronic acid]−. Quercetin pentoside (15) showed a deprotonated molecule [M-H]− at m/z 433.1094; however, quercetin aglycone (28) was confirmed by an ion peak at m/z 303.0966 [M+H]+ − of kaempferol neohesperidoside (16) was detected at m/z 593.1527. Isorhamnetin-O-hexoside (25) was detected by the presence of a protonated molecule [M+H]+ at m/z 479.1228, a production at m/z 317.0612 corresponding to [M+H-hexose]+, and a characteristic fragment at m/z 285.0323 319.1651 . Gossype6 [M+H]+ . Kaempfe285.0323 . The dep+ at m/z 273.0884 and a characteristic fragment at m/z 147.0214 [M+H-C6H6O3]+ [− at m/z 301.2041. Formononetin-O-hexoside (29) showed a protonated molecule [M+H]+ at m/z 431.1681 and a fragment at m/z 269.1243 [M+H-hexose]+. The deprotonated molecule [M-H]− of formononetin aglycone (37) was detected at m/z 267.0712 and yielded a fragment due to the methyl group loss at m/z 252.0420 [C-hexoside (18) showed a deprotonated molecule [M-H]− at m/z 415.1944, while daidzein aglycone (27) was detected at m/z 255.0551.Naringenin aglycone (36) showed a protonated molecule [M+H]C6H6O3]+ . Hespere+ at m/z 579.1859 and a fragment ion at m/z 561.1873 [M+H-H2O]+. Petunidin-O-hexoside (26) showed a protonated molecule [M+H]+ at m/z 479.0961 and a fragment ion at m/z 317.0662 [M+H-hexose]+. Malvidin-O-hexoside (33) was detected at m/z 491.1155 [M-H]− and confirmed by a fragment at m/z 329.0696 for [M-H-hexose]−.Procyanidin B2 (12) showed a protonated molecule [M+H]m/z 191.0349 [M-H]−, and daphnetin (46), which was confirmed by a peak at m/z 179.0861 [M+H]+ with a fragment at m/z 163.0503. Aldehydes were tentatively identified as hexenal (47), showing a protonated molecule [M+H]+ at m/z 99.0437 and a fragment ion at m/z 83.0493, while cinnamaldehyde (48) showed a protonated molecule [M+H]+ at m/z 133.1001 and a fragment ion at m/z 117.0684 + at m/z 415.3205, in accordance with published data [Tentatively identified coumarins were scopoletin (44), that was detected in both modes at 117.0684 . The chehed data ,29.2 as in succinic and malic acids  [The common neutral loss of 44 Daltons was observed in MS2 fragmentations of phenolic and organic acids due to the loss of COctively) . The ideIn the present study, MESC was evaluated for its hepatoprotective activity in paracetamol-induced liver toxicity. Plasma caspase 2 and cytokeratin 18 (CK18) were elevated in the group Gp II in comparison with the control group. Hofer et al. reported that caspase-cleaved cytokeratin 18 (CK18-Asp396) values were increased significantly in Gp II . Caspasep < 0.01) in plasma caspase 2 and CK18 levels, corresponding to 346.39% and 139.31% increases, respectively, as compared to the levels in Gp I, considering the values in the control group as 100% (Toxicity by paracetamol (1 g/kg) led to a significant elevation ( as 100% . Pretreap < 0.01) in liver CAT level by 50.37%, compared to the levels in Gp I , resulting in inflammation ,41. MESCp < 0.05) were observed (p < 0.05) in C/ebpα, HNF-1α, and sirtuin-1 gene expression and a significant decrease (p < 0.05) of liver miRNA-34a and miRNA-223 genes expression compared to the levels in Gp II. MESC administration showed a significant increase (p < 0.05) in the expression of liver C/ebpα, HNF-1α, and sirtuin-1 genes by 404.35%, 179.55%, and 223.64%, respectively, as well as a significant decrease (p < 0.05) of liver miRNA-34a and miRNA-223 genes expression by 46.27% and 40.49%, respectively, compared with the levels in Gp II. Paracetamol overdose could suppress C/ebpα gene expression in hepatocytes in vivo, which was accompanied by a significant accumulation of cytokines. MESC and silymarin protected rats against paracetamol-induced hepatotoxicity. Oxidative stress due to C/ebpα gene expression depletion and uncontrolled ROS is known to be the primary pathogenic mechanism of paracetamol-induced liver toxicity, as well as the main inhibitor of hepatocytes protective factors. Our metabolomics results revealed that MESC is rich in flavonoid compounds that exerted pronounced antioxidative effects against paracetamol-induced depletion of C/ebpα gene expression in the hepatocytes of Gp II rats [A significant decrease of the expression of HNF-1α, sirtuin-1, and C/ebpα genes by 55.10%, 61.22%, and 77%, respectively, as well as a significant increase of the expression of liver miRNA-34a and miRNA-223 genes by 447.27% and 344.79%, respectively, in Gp II rats as compared with Gp I rats  demonstrated normal hepatocytes with no fibrosis or inflammation a, while S. costus; which supports the hepatoprotective activity of this plant [S. lappa root extract in rats [S. costus root extract pretreatment.Our LC–MS/MS results showed that flavonoids were the major components of is plant . Coumariis plant . Tejaswi in rats ; moreove in rats ,57. ThisMethanol (HPLC grade), paracetamol (99%), silymarin (98.5%), and Tween 80 were purchased from Merk . Other reagents were of high analytical grade.Saussurea costus  Lipsch. roots were purchased from a local herbalist . Identification of the plant was performed by the Agriculture Research Center, Cairo, Egypt. In Soxhlet, powdered roots (1 kg) were extracted with methanol . The extract was left to cool, filtered, and then evaporated  [zerland) . The obtn-hexane (2 × 100 mL), filtered, and evaporated. The crude oil was investigated for its composition in a GC–MS system  equipped with a mass spectrometer detector (5977A) at the National Research Centre, Cairo, Egypt. The GC was equipped with an HP-5MS column. Powdered roots (100 g) were macerated in m/z. The characterization of compounds was performed by the generation of the candidate formula with a mass accuracy limit of 10 ppm and also considering Rt, MS2 data, databases, and reference literature [The MESC was analyzed at the Proteomics and Metabolomics unit of the Children’s Cancer Hospital Egypt 57357, Cairo, Egypt. The analysis was carried out using an Exion LC Triple TOF 5600+ system  operated at 40 °C and equipped with an X select HSS T3 C-18 column  and a precolumn . MESC (50 mg) was dissolved in solvent working solution (MilliQ water: methanol: acetonitrile–50:25:25), sonicated (10 min), then centrifugated . The stock sample (50 µL) was diluted with the working solvent (1000 µL). The phytoconstituents of MESC were analyzed using UPLC/T-TOF–MS/MS in both negative and positive modes . Samplesterature . g, 10 min). The clear supernatant was tested for CAT using a Cayman Chemical Company kit . The UV spectrophotometric method for measuring catalase activity is centered on monitoring the change in 240 nm absorbance at high concentrations of hydrogen peroxide (30 mM). TNF and IL-6 levels in the liver were measured using a microplate reader at 450 nm . An anti-IL-6 or an anti-TNF-α monoclonal antibody and a biotin-conjugated monoclonal anti-IL-6 or anti-TNF-α antibody that binds to IL-6 or TNF-α captured by the first antibody were used in an indirect sandwich enzyme-linked immunosorbent assay to evaluate IL-6 and TNF-α. Streptavidin–HRP was added to the wells, followed by a substrate solution reacting with HRP. After stopping the reaction with acid, the absorbance was measured.Adult male albino rats  were purchased from the National Cancer Institute, Cairo University, Giza, Egypt. Rats were provided water and standard diet ad libitum, observed daily, and kept in polypropylene cages under normal environmental conditions (22 ± 2 °C). The prophylactic potential of MESC against paracetamol hepatotoxicity was evaluated. Ethical approval was obtained by the ethic committee of the faculty of applied medical science . Animals and the treatment schedule (4 weeks) were as follows: Gp I  and Gp II (positive control); rats were orally given Tween 80  in saline daily; Gp III rats were orally treated with silymarin , daily . Gp IV r2, (1.5 mM), 0.2 mM of each dNTP, 0.4 μM of specific primers . RNA (1 μg) in reaction buffer was mixed with dithiothreitol (10 nmol/L), oligo (dT) primer (25 pg), 0.5 mmol/L of each deoxyribonucleoside triphosphate (dNTP), and 200 units superscript II Rnase H-Reverse Transcriptase. The reactions were kept at 42 °C for 2 min, 42 °C for 50 min, 70 °C for 15 min, and then chilled to 4 °C. The PCR reaction mixture consisted of PCR buffer, MgCl primers  for hepaThe liver pieces were fixed in formaldehyde solution (10%) and then were examined for histopathological changes.p < 0.05 was considered statistically significant). Statistical analyses were carried out using GraphPad Prism . ANOVA with posttest Tukey’s multiple comparisons were performed. Values (n = 10) are presented as mean ± standard deviation for ELISA measurements and PCR analyses of gene expression  were characterized as belonging to different chemical classes, flavonoids being the major constituents besides organic acids, coumarins, and anthocyanidin. S. costus has a potent hepatoprotective activity through the modulation of cellular cytokines release and miRNA-34a and miRNA-223 expression. S. costus reduced the levels of caspase 2 and CK18, TNF-α, and IL-6 and increased catalase activity. Furthermore, it increased HNF-1α, sirtuin-1, and C/ebpα expression and decreased miRNA-34a and miRNA-223 gene expression. S. costus minimized some negative symptoms and pathological changes. We hope that this study will attract attention towards this plant myriad chemical constituents and its great potential in health care.Herein for the first time, we were able to identify the chemical profile of


Worse sleep quality and increased inflammatory markers in women with schizophrenia (Sch) have been reported . However, the physiological mechanisms underlying the interplay between sleep and the inflammatory pathways are not yet well understood .Analyze the relationship between Neutrophil/Lymphocyte (NLR), Monocyte/Lymphocyte (MLR) and Platelet/Lymphocyte (PLR) ratios, and insomnia in Sch stratified by sex.Final sample included 176 Sch patients (ICD-10 criteria) . Assessment: PANSS, Calgary Depression Scale (CDSS), and Oviedo Sleep Questionnaire (OSQ) to identify a comorbid diagnosis of insomnia based on ICD-10. Fasting counting blood cell were performed to calculate ratios. Statistics: U Mann-Whitney, logistic regression.Insomnia as comorbid diagnosis was present in 22 Sch (12.5%) with no differences between sex , neither in their age. Female patients with insomnia showed increased NLR . However, no differences in PLR and MLR were found, neither in any ratio in males. Regression models using insomnia as dependent variable and covariates  were estimated. Females: presence of insomnia was associated with NLR [OR=3.564 (p=0.032)], PANSS-positive [OR=1.263 (p=0.013)] and CDSS [OR=1.198 (p=0.092)]. Males: only PANSS-positive [OR=1.123 (p=0.027)] and CDSS scores [OR=1.220 (p=0.005)] were associated with insomnia.NLR represent an inflammatory marker of insomnia in Sch but only in female patients. Improving sleep quality in these patients could help to decrease their inflammatory response.No significant relationships.


Non-polio Enteroviruses (EV) are important neonatal CNS pathogens. Multiple EV genotypes have been detected in pediatric CSF, e.g. Echovirus (E) 6 and E30. CSF innate immune responses to EV genotypes remain poorly defined. Most data are from EV-A71 or E30 CNS infections and do not compare responses between these or other EV types. We sought to better define innate immune responses to EV genotypes in CSF.Salvaged standard of care CSF samples from ≤6 month olds  or EV PCR(-) controls) from Jan 2010 - Dec 2020 were tested in duplicate on a 21- cytokine bead panel (MilliporeSigma). EV positive samples were previously genotyped by sequencing the viral capsid gene. Cytokine levels calculated from the standard curve were compared by Kruskal-Wallis and post-hoc analysis (GraphPad Prism 8.4.3). Natural partitioning of participants was explored using principal component analysis and cluster analysis (IBM SPSS v27). The utility of cytokine signatures in predicting EV status was explored using discriminant analysis and ROC analysis (IBM SPSS v27).Data from 72 CSF with E6 (N=16), E9 (N= 9), E18 (N=9), and E30 (N=21) showed significant differences among EV genotypes vs. controls for 20 cytokines (IL-17 was excluded). Significant differences in cytokine levels in EV CSF vs controls were seen: E6 for all 20 cytokines; E9 for Fractalkine, IP10, and MCP1; E18 for Fractalkine and MCP1; E30 19 cytokines (not GM-CSF). PCA revealed only minor overlap of controls and EV positives; EV types overlapped, except E30, differing most from E9 and E18 but overlapping E6. The most important type-differentiating cytokines by PCA were MCP1, Fractalkine, IL-8, and IL-10. Patterns in DA resembled PCA; controls clearly separated from EV CSF, E30 being the most distinct. Overall, the discriminant model correctly classified EV type or controls at a 63% rate - highest for controls (94.1%) and E30 (74.1%). In the DA model, the most important cytokines were IP-10, IL-2, IL-1Ra, and Fractalkine. Discriminant scores had the largest area under the curve (AUC), 0.990. Among cytokines, IFNa2 had the largest AUC, 0.987.Preliminary data show significant EV-genotype differences in innate immune response to CNS infections. Cytokine patterns may serve as a key predictor for discerning EV genotypes.Brian R. Lee, PhD, MPH, CDC: Grant/Research Support|Merck: Grant/Research Support Christopher J Harrison, MD, Astellas: Grant/Research Support|GSK: Grant/Research Support|Merck: Grant/Research Support|Pediatric news: Honoraria|Pfizer: Grant/Research Support Rangaraj Selvarangan, BVSc, PhD, D(ABMM), FIDSA, F(AAM), BioFire: Grant/Research Support|Luminex: Grant/Research Support.


A vascular system in plants is a product of aromorphosis that enabled them to colonize land because it delivers water, mineral and organic compounds to plant organs and provides effective communications between organs and mechanical support. Vascular system development is a common object of fundamental research in plant development biology. In the model plant Arabidopsis thaliana, early stages of vascular tissue formation in the root are a bright example of the self-organization of a bisymmetric (having two planes of symmetry) pattern of hormone distribution, which determines vascular cell fates. In the root, vascular tissue development comprises four stages: (1) specification of progenitor cells for the provascular meristem in early embryonic stages, (2) the growth and patterning of the embryo provascular meristem, (3) postembryonic maintenance of the cell identity in the vascular tissue initials within the root apical meristem, and (4) differentiation of their descendants. Although the anatomical details of A. thaliana root vasculature development have long been known and described in detail, our knowledge of the underlying molecular and genetic mechanisms remains limited. In recent years, several important advances have been made, shedding light on the regulation of the earliest events in provascular cells specification. In this review, we summarize the latest data on the molecular and genetic mechanisms of vascular tissue patterning in A. thaliana root. The first part of the review describes the root vasculature ontogeny, and the second reconstructs the sequenceof regulatory events that underlie this histogenesis and determine the development of the progenitors of the vascularinitials in the embryo and organization of vascular initials in the seedling root. Evolutionary formation of a vascular system in plants wasa necessary prerequisite for terrestrial colonization . Vasculature provides mechanical support, effectivetransportation of water, and mineral and organic compoundsas well as signal molecules and by this has enabled plants toreach enormous sizes and populate different territories. Thevascular system consists of two domains different in theirstructure and functions. These are xylem that provides watertransportation and delivers mineral compounds from the rootto above-ground organs; and phloem that conveys organiccompounds from photosynthesizing tissues rootward .In angiosperms, the mature xylem consists of (1) watertransportationvessels; (2) fibers to provide mechanical support;(3) parenchyma cells . The vesselsare the hollow tubes formed by the cells connected in a rawand having perforations in the anticlinal walls and pores in thepericlinal walls . The vessels and fibers are a productof the programmed death of the cells that have formed a lignifiedsecondary cell wall . Meanwhile, the living cellsof parenchyma perform a storage function, participating invessel lignification and regulating the water transport speed.The phloem, on the other hand, consists of (1) sieve tubesto transport organic substances; (2) companion cells; (3) fibersand sclereids to provide mechanical support, and (4) parenchymacells . Unlikethe lignified hollow vessels of the xylem, the sieve tubes area strand of living cells (sieve elements) communicating bysieve fields, anticlinal-wall regions with high numbers ofsmall pores. The sieve elements form a thickened non-lignifiedsecondary cell wall  and their main feature isthe lack most of the organelles including a nucleus, vacuole,rough endoplasmic reticulum, Golgi body, cytoskeleton,ribosomes whose presence could prevent substances transportation.The viability of the sieve elements is maintainedby companion cells – the parenchyma cells with large nucleiand mitochondria, directly contacting sieve elements. As for mechanical phloem elements – fibers and sclereids – they differfrom each other by the shape of their cells. While the formerare strongly elongated and pointed at the ends, the latter arejust slightly elongated.Organization of vascular system is different for differentorgans in different plant species at different stages of theirdevelopment . Nevertheless, the mechanisms determiningits developmentare quite conservative . Plant cells are not capable of migration, so duringmorphogenesis, the tissue and organ architecture is formedby regulating the sequence and orientation of cell divisions.In terms of its anatomy, the vascular system development hasbeen described in much detail , however, the molecular andgenetic mechanisms responsible for this process are muchless known. Our current understanding of these mechanismsis mainly based on the investigation of the model plant ArabidopsisthalianaIn the further sections of this review, we will provide a shortdescription of vascular tissue histogenesis in this plant speciesand reconstruct the corresponding sequence of regulatoryevents. We will describe the control of root vascular systemdevelopment in the embryo and seedling, i. e. the earlieststages of its formation. As for the mechanisms controllingvasculature development at later stages, their description canbe found in the recent reviews .GlossaryAmphicribral vascular bundle – a vascular bundle inwhich the phloem surrounds the xylem.Anticlinal – located in a plane perpendicular to the surfaceof a tissue or organ. Talking about anticlinal cell wallsor divisions we will mean an anticlinal plane perpendicularto the central axis of an organ.Anticlinal cell division – cell division in the anticlinalplane that leads to an increase in length.Asymmetric cell division – results in the formation oftwo daughter cells with different cell fates.Cortex – a cell layer surrounding the endodermis.Diarch vascular bundle – a vascular bundle whose phloemand xylem are located at different radii, wherein tworays of xylem are distinguished.Endodermis – the innermost cell layer surrounding thestele.Hypophysis is the upper cell of the suspensor, which acquiresits identity at the 16–32 cell stage; gives rise to thequiescent center  and the root cap.Periclinal – located in a plane parallel to the surface ofa tissue or organ.Periclinal cell division – cell division in the periclinalplain leading to an increase in the number of cell layersin the radial direction.Pericycle – parenchyma cell layer surrounding conductivetissues and forming the stele outer layer.Primary meristem – formed during embryogenesis.Procambium – indeterminate primary vascular meristemcells located between the xylem plate and phloempoles in the root of Arabidopsis thaliana.Provascular initials – four proembryo cells occurring atthe early globular stage to form the entire provascularmeristem of the root/hypocotyl, and only it.Provascular root/hypocotyl meristem – primary meristemfrom which the primary vascular system of theseorgans differentiates after embryo germination.Root apical meristem – primary root meristem to produceall cells of the root during its post-embryonicgrowth.Secondary meristem – formed during the postembryonicperiod.Stele  – primary conductive tissues locatedin the center of the axial organ, and surrounded bya pericycle.Suspensor – a structure at the base of an embryo thatconnects it to endosperm and consists of the descendantsof a two-celled pro-embryo basal cell.Vascular cambium – secondary vascular meristem toprovide root thickening.Xylem plate – a layer of primary xylem cells (or theirpredetermined precursors) located in the central planealong the root axisThere are primary (produced by the primary meristem) andsecondary (produced by the secondary meristem) vasculartissues.Development of root primary conductive tissuesAt the globule stage of A. thaliana embryogenesis the specificationof four provascular initials occurs. Provascular initialsundergo oriented divisions, finally giving rise to the provascularmeristem of the embryonic root and hypocotyl . Thecells of provascular meristem are not yet differentiated, but thecellular fate of some of them has already been determined –after the embryo germination they give birth either to xylemor to phloem cells. The positions of these predetermined cellsin the provascular meristem matches that of the bisymmetric diarch organizationof the vascular system in the postembryonic root tip: in itstransverse – section, there is one layer of xylem precursor cellssurrounded on both sides by procambial cells that separate thefuture xylem from two files of phloem progenitor cells, whichlie in a perpendicular plane  .This structure is surrounded by pericycle cells that are alsoderived from provascular initials, so together they form a centralcylinder or a stele . It is noteworthy that theterminology designating the cells in developing root vascularsystem is rather blurred . In particular, theterm ‘procambium’ is applied to address either indeterminate cells of the primary vascular tissue in seedlings (and theirprogenitors) or the whole embryonic provascular meristem.Soon after germination, vascular elements start to differentiatein a hypocotyl stele and cotyledon veins, the provascularmeristem of the latter comes from the shoot apical meristem. From the hypocotyl, the processspreads upwards and downwards taking the epicotyl and root,respectively . In A. thaliana, these are the protophloemsieve elements adjacent to the pericycle that differentiatefirst, and since the cells surrounding them keep elongating,protophloem cells soon die to be functionally replaced bythe metaphloem sieve elements placed closer to the center ofthe stele . Later, theprotoxylem vascular elements are formed that are located atthe poles of the xylem plate and have annular or spiral thickeningsof the secondary cell walls. The last cells to differentiateare the metaxylem cells occupying the central position in thexylem plate and having pitted or reticulate lignin deposits.While the root grows in length, its new cells are producedthrough anticlinal division of the cells in the apical meristemlocated at the root tip . In A. thaliana,the root apical meristem is closed, i. e. different stem cells can produce not any but strictly limited set of celltypes and for each differentiated cell it is easy to trace whichstem cell it has originated from . Among the steleinitials those can be distinguished that give birth to (1) protoxylem;(2) metaxylem; (3) procambium and sieve elementsof proto- and metaphloem ;(4) only procambial cells; (5) pericycle . The mutual arrangement of initials correspondsto the diarch organization of young root vasculature, so thecell identity established in the embryo provascular meristem ismaintained in the root apical meristem. Here it is worth mentioningthat apart from the proto- and metaxylem, proto- andmetaphloem and procambium there are also companion cells.Some authors designate them more srictly as companion-likecells . These cells are adjacent to the sieveelements of proto- and metaphloem and possess a number ofmorphological and physiological characteristics of companioncells  but, unlike the latter, theydo not share a common initial with the proto- and metaphloemelements in the stem cell niche . Thecompanion-like cells differentiate when the protophloemsieve elements start functioning . InA. thaliana, the xylem and phloem parenchyma, fibers andtrue companion cells differentiate only during the secondarygrowth .In A. thaliana primary vascular system, periclinal divisionsof procambium cells are few, but after differentiation of theprimary vascular elements, these cells begin to actively dividepericlinally. The periclinal divisions also occur in the pericycle cells adjacent to the xylem plate. As a result, a closed cell ringforms around the xylem to give birth to the vascular cambium . It is noteworthythat only those procambium and pericycle cells in direct contactwith the xylem primary vessels give rise to the vascularcambium, i. e., have the properties of stem cells  while the descendants of other proliferating procambialcells differentiate into the phloem.Thus, the diarch root vasculature transforms into amphicribralone, in which the xylem is surrounded by thephloem with the cambium placed in between .Through asymmetric division, every initial is capable of producingphloem cells outwards and xylem cells inwards, sothe root gets thicker . In some species,e. g., in the vast majority of monocots, the cambium is notformed and no secondary growth is initiated. In this case, allprocambium cells get differentiated.The development of a multicellular organism is accompaniedby a gradual increase in the limitation of cellular potencies. Atthe first stage of this process predetermination or specificationoccurs, in other words, the fate of a totipotent cell is establishedin terms of the progenitor of what type of cells it will become.Meanwhile, the cell remains undifferentiated and can changeits fate under certain conditions. The process of cell identitydetermination involves the local accumulation of signalmolecules, which either activate or suppress the activity thegene networks inherent in specific cell types. In this case, animportant role is given to the non-cell-autonomous factors ableto move between cells and form gradients .Provascular stem cells specification at the early globularstage of embryogenesis is preceded by a series of cell divisionsand embryo polarity determination . The proper accomplishment of these processes isessential for the vascular tissue to begin its development fromthe right number of cells placed in the right positions. Planthormone auxin is a key regulator of embryogenesis, whoseheterogeneous distribution provides positional information,which directs embryo development . The main auxineffector in embryogenesis is transcription factor (TF) AUXINRESPONSE FACTOR 5 (ARF5)/MONOPTEROS (MP) and it is believedthat forming the auxin signal-distribution pattern is providedmainly due to feedbacks in regulation of phytohormonebiosynthesis, its polar intercellular transport and signalingpathway . As a result, at the early stagesof embryogenesis, auxin is accumulated in the apical cells todetermine the embryo polarity . Startingfrom the early globular stage (32 cells), its maximum is shiftedto the upper cells of the suspensor including the hypophysisthat later gives rise the quiescent center of the root apicalmeristem .Although the four provascular initials are only distinguishedat the early globular stage , the cellular identity of vascular tissue progenitors is determined in the fourinner cells of the lower layer of the proembryo as early as atdermatogen stage  . Via periclinaldivision at transferring to the 32-cell stage, they produceoutwards the ground tissue progenitors that lose the vascularidentity of their maternal cells  . A necessary condition for provascular-initial specification is ARF5/MP-dependent activation ofthe auxin signaling pathway, but meeting this condition aloneis not enough . Whileparticular auxin assistants remain unknown, its is suggestedthat this role is performed not by a single key regulator butby a multicomponent regulatory network, and TF G-BOXBINDING FACTOR 2 (GBF2) is believed to be one of itsmembers  . GBF2 is assumedto modulate ARF5/MP binding to target-gene promoters. It isworth mentioning here that the state, in which vascular systemprogenitors are uniformly specified is most likely transientwith no stable uniform cellular identity.As the oriented divisions of the provascular initials and theirdescendants continue, the hypocotyl and root vascular systemsbecome patterned through specification of particularcellular types. An important aspect at this stage is setting theboundaries for the cellular domains with different structuraland functional identities. By the end of embryogenesis, inthe embryo provascular meristem, the cell identity of all elementssuch as proto- and metaphloem, proto- and metaxylem,companion-like cells and procambium has been determinedas evidenced by the data on cell morphologyand expressionof marker genes  .In A. thaliana, the bisymmetry of the future root is believedto be predetermined already at the early globular stage by theextended contact between two provascular initials locateddiagonally relative to each other . This contactis probably formed due to the inaccurate match of cell divisionplanes in proembryo (at the four-cell stage) and is importantfor xylem plate formation . Startingfrom the early heart stage, auxin begins to be activelytransported into such contacting provascular cells from thecotyledon primordia located above them, while in other cellsthe hormone levels remain low . The local increase inauxin concentration is necessary for the specification of xylemprogenitor cells .At the same time, the cells rich in auxin begin to act as anorganizing center for the provascular meristem, coordinatingits growth through periclinal divisions and establishment ofbisymmetric organization . Auxin inducesthe ARF5/MP-dependent expression of TFs TARGETOF MONOPTEROS 5 (TMO5) and TMO5-LIKE1 (T5L1), which,forming heterodimers with the auxin-independent LONESOME HIGHWAY (LHW) TF , activatethe expression of cytokinin biosynthesis genes LONELYGUY3 (LOG3) and LOG4  . Simultaneously, auxin blockscytokinin signal transduction, increasing the expression ofgene ARABIDOPSIS HISTIDINE PHOSPHOTRANSFERPROTEIN 6 (AHP6 ) encoding a cytokinin signaling pathwayinhibitor , soa local cytokinin source is formed in xylem progenitors lackingcytokinin signalingThe high cytokinin level, on the one hand, limits auxin effluxfrom xylem progenitor cells by controlling the localizationof auxin transporter PIN-FORMED 1 (PIN1) on the cell membrane. On theother hand, cytokinin diffuses into neighboring cells followingthe concentration gradient. In these cells, in the absence of theinhibitor , cytokinin activates signalingcascade to stimulate periclinal divisions .Simultaneously, cytokinin signaling suppresses cell specificationinto xylem . This mechanismprovides for the radial growth of the provascular meristem,which is accompanied by spatial separation of the domainsfor increased auxin signal (cells obtain xylem identity) andcytokinin signal . Its sufficiencyfor self-organization of the bisymmetric pattern was confirmedusing a mathematical model .In early embryogenesis, provascular-meristem progenitorsbegin to express genes encoding peptide hormone CLAVATA 3(CLV3)/EMBRYO SURROUNDING REGION 25 (CLE25)and mobile TFs of the DNA BINDING WITH ONE FINGER(DOF) family united in the PHLOEM EARLY DOF (PEAR)group marking sieve-element progenitors in the postembryonicperiod . CLE25is expressed starting from a 64-cell embryo stage . Cytokinin-independent expression of PEAR1 isdetected already at a 16-cell stage, and starting from an earlyheart stage, this gene expression is activated by cytokinin. It is assumed that the CLE25 peptidebinding to the CLE-RESISTANT RECEPTOR KINASE(CLERK)-CLV2 receptor together with the PEAR1 TF contributeto the early specification of phloem progenitor cells.However, unlike that for xylem, the mechanism to initiatephloem development in embryogenesis remains unknown.Bisymmetric pattern in steleIn the postembryonic period, the stele cells progenitors maintainthe bisymmetric pattern established in embryogenesis,so some of the mechanisms regulating the cell dynamics andvascular-system element predetermination in provascular meristemkeep functioning even after germination. However, itcannot be said with complete certainty that these mechanismsare identical.In the apical meristem, auxin-rich xylem progenitors retainthe function of an organizing center, carrying out TMO5/LHW-mediated regulation of cytokinin levels in procambialcells  . The high contentof active cytokinin in xylem cells is maintained by TMO5/LHW-dependent activation of not only cytokinin biosynthesisgenes LOG3 and LOG4 but also of the BGLU44 gene encodinga β-glucosidase enzyme . Cytokinin response inxylem is blocked by auxin through AHP6 gene expression induction  as well as through limitingthe activity of TMO5/LHW by activating the ACAULIS 5(ACL5)–SUPPRESSOR OF ACAULIS5 LIKE3 (SACL3)regulatory module blocking the formation of the TMO5/LHWheterodimer by competing with TMO5 for binding to LHW . Meanwhile,in xylem-adjacent procambial cells, the level of thecytokinin diffusing from the xylem is limited by TMO5/ LHWdependentactivation of CYTOKININ OXIDASE 3 (CKX3).The activation is mediated by the mobile SHORT ROOT(SHR) TF, encoded by TMO5/LHW target gene. The combinedaction of multidirectional regulatory modules ensuresthe stability of the pattern to short-term fluctuations in auxinconcentrations in xylem cells, while maintaining its sensitivityto slower/stable changes . What is interestingis that the SHR gene is important not only for the rootradial symmetry but also for the functioning of the quiescentcenter .TMO5/LHW-induced cytokinin activates the transcriptionof the DOF2.1 TF in the procambial cells surrounding thexylem pole, thus controlling their division  . It is worth noting that, besides xylem cells, it isdifferentiated phloem that transports the phytohormone andthus can be a source of cytokinin in the root apical meristem. However, mathematical modelinghas demonstrated that phloem cytokinin is not a fundamentalsource of the positional information for bisymmetric patternformation . At the same time, the highcytokinin content at the phloem poles arranges periclinaldivisions of procambium cells through activating the genesof mobile TFs of the DOF family united in the PEAR groupincluding PEAR1, PEAR2, TMO6, DOF6 . They create a concentration gradientand activate the periclinal divisions of the procambial cells surroundingthe phloem pole. HOMEODOMAIN LEU-ZIPPERclass-III (HD-ZIP III), TFs whose expression domain is set inthe central part of the stele (see below) limit the activity of thePEAR TFs , and PEAR1 activates the transcriptionof the genes belonging to the HD-ZIP III family, forminga negative feedback loop.Proto- and metaxylem predeterminationAs in embryogenesis, auxin is necessary for xylem cellspredeterminationin the root apical meristem. In proto- andmetaxylem predetermination, a key role is given to theSHR and miRNA165/166 mobile regulators . SHRis produced by xylem cells, from where the TF spreadstowards the periphery and, upon reaching the endodermis,activates the SCARECROW (SCR) TF, so they togetherinduce miRNA165/166 expression . MicroRNAs diffuse into neighboringcells, creating a concentration gradient towards the center ofthe root. In the stele, miRNA165/166 suppress the expressionof the genes encoding the TFs of the HD-ZIP III family, limitingit to the central domain . In such a way, themetaxylem cells are predetermined. Whether this mechanismworks in embryogenesis remains unknown, but this is a possibilitysince the PHABULOSA (PHB) TF of the HD-ZIP IIIfamily is expressed in the embryo root .Predetermination of phloem elementsThe phloem markers expressed in progenitors and induce thetissue development include a number of the DOF family TFs; strigolactonesignaling pathway suppressors SUPPRESSOR OF MAX21-LIKE 3 (SMLX3), SMLX4 and SMLX5 ; membrane proteins BREVIS RADIX (BRX), OCTOPUS(OPS), OPS-LIKE 2 (OPL2) ;phosphatase COTYLEDON VASCULAR PATTERN 2(CVP2) and its homolog CVP2-LIKE 1 (CVL1) ; the ALTERED PHLOEM DEVELOPMENT(APL) TF .The formation of protophloem elements is controlled byshifting the balance towards inducing or suppressing mechanismswith the central link connecting the opposing regulatorymodules being phloem-specific TFs of the DOF family. On the one hand, these TFs induce theexpression of phloem development activators, such as APLas well as their own genes, forming a positive feedback loop.On the other hand, DOFs induce the expression of CLE25,CLE26, and CLE45 signaling peptides migrating to neighboringcells where they trigger an inhibitory regulatory module. Interacting with the BARELY ANY MERISTEM(BAM) receptors and the CLAVATA3 INSENSITIVERECEPTOR KINASE (CIK) co-receptors, the CLE peptidesinduce the degradation of the DOF family TFs, suppressingthe formation of protophloem elements. The activity of theCLE peptide receptors can be additionally regulated, e. g., bythe MEMBRANE-ASSOCIATED KINASE REGULATOR 5(MAKR5)  or CORYNE (CRN)  regulators. The TFs of the DOF family activatethe expression of the genes encoding the OPS membraneprotein suppressing the BAM-CIK module .Properly positioned protophloem progenitor cells overcomethe inhibitory effect of CLE peptides due to the DOF TF accumulationdetermined by the positive feedback. Such a balancingmechanism makes it possible to repattern the phloemin case protophloem development has been disrupted . Here it should be noted that metaphloem developmentis probably regulated by other mechanisms and does notdepend on that of the protophloem .During phloem formation, the phloem/procambium stemcell divides anticlinally to produce a daughter procambiumand sieve-element progenitor to divide periclinally and forma procambium progenitor and a phloem sieve-element progenitor.The latter undergoes another periclinal division toproduce proto- and metaphloem progenitors . Companion-like cells are another product ofasymmetric division, but come from a different initial. Theseasymmetric cell divisions are controlled by a positional signal,a SHR-protein gradient whose migration into the endodermisactivates miRNA165/166 and induces asymmetric divisionsproducing companion-like cells, while SHR movement intothe phloem is necessary for the asymmetric divisions leadingto proto- and metaxylem formation .The vascular system of A. thaliana root is set at the earlieststages of embryogenesis. Wherein, the predetermination ofprovascular initials implies a labile, unstable, and reversiblespecification based on the physical arrangement of cells in theembryo and influenced by a complex regulatory network oftranscription factors. An interesting moment here is that bothxylem  and phloem  markers are jointly expressed by provascular initials inearly embryogenesis, but later they are separated into differentspatial domains in the provascular meristem and seedling.In A. thaliana, the vascular system is patterned by the timeof embryo maturation. Partially, the gene network that controlsthis process in embryogenesis continues to maintain thevascular system structure of the growing root of the seedlingand later during plant ontogenesis. This is associated withlocal accumulation of the molecular markers that are stablyexpressed in progenitor cells of a certain type. However,the factors working both in embryogenesis and during postembryonicdevelopment can act at these stages in differentwaysDespite the significant progress that has recently beenachieved in understanding the molecular and genetic mechanismsregulating vascular system development in plants,many questions remain open, in particular, those related tothe existence of parallel regulatory pathways and feedforwardloops. This is a good basis for building mathematicalmodels whose analysis helps shed light on the relationshipbetween various regulatory circuits and their functionalsignificance.The authors declare no conflict of interest.Agustí J., Blázquez M.A. Plant vascular development: mechanisms andenvironmental regulation. Cell. Mol. Life Sci. 2020;77(19):3711-3728. DOI 10.1007/s00018-020-03496-w.Bauby H., Divol F., Truernit E., Grandjean O., Palauqui J.C. Protophloemdifferentiation in early Arabidopsis thaliana development.Plant Cell Physiol. 2007;48(1):97-109. DOI 10.1093/pcp/pcl045.Baum S.F., Dubrovsky J.G., Rost T.L. Apical organization and maturationof the cortex and vascular cylinder in Arabidopsis thaliana(Brassicaceae) roots. Am. J. Bot. 2002;89(6):908-920. DOI 10.3732/ajb.89.6.908Bishopp A., Help H., El-Showk S., Weijers D., Scheres B., Friml J.,Benková E., Mähönen A.P., Helariutta Y. A mutually inhibitory interactionbetween auxin and cytokinin specifies vascular pattern inroots. Curr. Biol. 2011a;21(11):917-926. DOI 10.1016/j.cub.2011.04.017.Bishopp A., Lehesranta S., Vatén A., Help H., El-Showk S., Scheres B.,Helariutta K., Mähönen A.P., Sakakibara H., Helariutta Y. Phloemtransportedcytokinin regulates polar auxin transport and maintainsvascular pattern in the root meristem. Curr. Biol. 2011b;21(11):927-932. DOI 10.1016/j.cub.2011.04.049.Bonke M., Thitamadee S., Mähönen A.P., Hauser M.T., Helariutta Y.APL regulates vascular tissue identity in Arabidopsis. Nature.2003;426(6963):181-186. DOI 10.1038/nature02100.Busse J.S., Evert R.F. Pattern of differentiation of the first vascular elementsin the embryo and seedling of Arabidopsis thaliana. Int. J.Plant Sci. 1999;160(1):1-13. DOI 10.1086/314098.Cai Q., Fukushima H., Yamamoto M., Ishii N., Sakamoto T., Kurata T.,Motose H., Takahashi T. The SAC51 family plays a central rolein thermospermine responses in Arabidopsis. Plant Cell Physiol.2016;57(8):1583-1592. DOI 10.1093/pcp/pcw113.Carlsbecker A., Lee J.Y., Roberts C.J., Dettmer J., Lehesranta S.,Zhou J., Lindgren O., Moreno-Risueno M.A., Vatén A., ThitamadeeS., Campilho A., Sebastian J., Bowman J.L., Helariutta Y.,Benfey P.N. Cell signalling by microRNA165/6 directs gene dosedependentroot cell fate. Nature. 2010;465(7296):316-321. DOI10.1038/nature08977.Cheng C.Y., Kieber J.J. Cytokinin signaling in plants. In: Howell S.(Ed.) Molecular Biology. The Plant Sciences. Vol. 2. New York:Springer, 2014;269-289. DOI 10.1007/978-1-4614-7570-5_14.Courtois-Moreau C.L., Pesquet E., Sjödin A., Muñiz L., Bollhöner B.,Kaneda M., Samuels L., Jansson S., Tuominen H. A unique programfor cell death in xylem fibers of Populus stem. Plant J. 2009;58(2):260-274. DOI 10.1111/j.1365-313X.2008.03777.x.De Rybel B., Adibi M., Breda A.S., Wendrich J.R., Smit M.E.,Novák O., Yamaguchi N., Yoshida S., Van Isterdael G., Palovaara J.,Nijsse B., Boekschoten M.V., Hooiveld G., Beeckman T., WagnerD., Ljung K., Fleck C., Weijers D. Integration of growth andpatterning during vascular tissue formation in Arabidopsis. Science.2014a;345(6197):1255215. DOI 10.1126/science.1255215.De Rybel B., Breda A.S., Weijers D. Prenatal plumbing – vascular tissueformation in the plant embryo. Physiol. Plant. 2014b;151(2):126-133. DOI 10.1111/ppl.12091.De Rybel B., Mähönen A.P., Helariutta Y., Weijers D. Plant vasculardevelopment: from early specification to differentiation. Nat. Rev.Mol. Cell Biol. 2016;17(1):30-40. DOU 10.1038/nrm.2015.6.De Rybel B., Möller B., Yoshida S., Grabowicz I., Barbier de Reuille P.,Boeren S., Smith R.S., Borst J.W., Weijers D. A bHLH complexcontrols embryonic vascular tissue establishment and indeterminategrowth in Arabidopsis. Dev. Cell. 2013;24(4):426-437. DOI10.1016/j.devcel.2012.12.013.Desvoyes B., Echevarría C., Gutierrez C. A perspective on cell proliferationkinetics in the root apical meristem. J. Exp. Bot. 2021;72(19):6708-6715. DOI 10.1093/jxb/erab303.Dolan L., Janmaat K., Willemsen V., Linstead P., Poethig S., RobertsK.,Scheres B. Cellular organisation of the Arabidopsis thaliana root.Development. 1993;119(1):71-84. DOI 10.1242/dev.119.1.71Evert R.F., Eichhorn S.E. Esau’s Plant Anatomy. Meristems, Cells, andTissues of the Plant Body: Their Structure, Function, and Development.New Jersey: Wiley, 2006. DOI 10.1002/0470047380.Friml J., Vieten A., Sauer M., Weijers D., Schwarz H., Hamann T., OffringaR., Jürgens G. Efflux-dependent auxin gradients establish the apical-basal axis of Arabidopsis. Nature. 2003;426(6963):147-153.DOI 10.1038/nature02085.Furuta K.M., Hellmann E., Helariutta Y. Molecular control of cell specificationand cell differentiation during procambial development.Annu. Rev. Plant. Biol. 2014;65:607-638. DOI 10.1146/annurevarplant-050213-040306.Graeff M., Hardtke C.S. Metaphloem development in the Arabidopsisroot tip. Development. 2021;148(18):dev199766. DOI 10.1242/dev.199766.Grigg S.P., Galinha C., Kornet N., Canales C., Scheres B., Tsiantis M.Repression of apical homeobox genes is required for embryonic rootdevelopment in Arabidopsis. Curr. Biol. 2009;19(17):1485-1490.DOI 10.1016/j.cub.2009.06.070.Gujas B., Kastanaki E., Sturchler A., Cruz T.M.D., Ruiz-Sola M.A.,Dreos R., Eicke S., Truernit E., Rodriguez-Villalon A. A reservoir ofpluripotent phloem cells safeguards the linear developmental trajectoryof protophloem sieve elements. Curr. Biol. 2020;30(5):755-766.DOI 10.1016/j.cub.2019.12.043.Hazak O., Brandt B., Cattaneo P., Santiago J., Rodriguez-Villalon A.,Hothorn M., Hardtke C.S. Perception of root-active CLE peptidesrequires CORYNE function in the phloem vasculature. EMBO Rep.2017;18(8):1367-1381. DOI 10.15252/embr.201643535.Help H., Mähönen A.P., Helariutta Y., Bishopp A. Bisymmetry in theembryonic root is dependent on cotyledon number and position.Plant Signal. Behav. 2011;6(11):1837-1840. DOI 10.4161/psb.6.11.17600.Heo J.O., Roszak P., Furuta K.M., Helariutta Y. Phloem development:current knowledge and future perspectives. Am. J. Bot. 2014;101(9):1393-1402. DOI 10.3732/ajb.1400197.Kang Y.H., Hardtke C.S. Arabidopsis MAKR5 is a positive effector ofBAM3-dependent CLE45 signaling. EMBO Rep. 2016;17(8):1145-1154. DOI 10.15252/embr.201642450.Katayama H., Iwamoto K., Kariya Y., Asakawa T., Kan T., Fukuda H.,Ohashi-Ito K. A negative feedback loop controlling bHLH complexesis involved in vascular cell division and differentiation inthe root apical meristem. Curr. Biol. 2015;25(23):3144-3150. DOI10.1016/j.cub.2015.10.051.Kim H., Zhou J., Kumar D., Jang G., Ryu K.H., Sebastian J., MiyashimaS., Helariutta Y., Lee J.Y. SHORTROOT-mediated intercellularsignals coordinate phloem development in Arabidopsis roots. PlantCell. 2020;32(5):1519-1535. DOI 10.1105/tpc.19.00455.Kuroha T., Tokunaga H., Kojima M., Ueda N., Ishida T., Nagawa S.,Fukuda H., Sugimoto K., Sakakibara H. Functional analyses ofLONELY GUY cytokinin-activating enzymes reveal the importanceof the direct activation pathway in Arabidopsis. Plant Cell. 2009;21(10):3152-3169. DOI 10.1105/tpc.109.068676.Lau S., De Smet I., Kolb M., Meinhardt H., Jürgens G. Auxin triggersa genetic switch. Nat. Cell Biol. 2011;13(5):611-615. DOI 10.1038/ncb2212Lau S., Slane D., Herud O., Kong J., Jürgens G. Early embryogenesisin flowering plants: setting up the basic body pattern. Annu. Rev.Plant Biol. 2012;63:483-506. DOI 10.1146/annurev-arplant-042811-105507.Li X., Wu H.X., Southerton S.G. Comparative genomics reveals conservativeevolution of the xylem transcriptome in vascular plants.BMC Evol. Biol. 2010;10:190. DOI 10.1186/1471-2148-10-190Lucas W.J., Groover A., Lichtenberger R., Furuta K., Yadav S.R., HelariuttaY., He X.Q., Fukuda H., Kang J., Brady S.M., Patrick J.W.,Sperry J., Yoshida A., López-Millán A.F., Grusak M.A., Kachroo P.The plant vascular system: evolution, development and functions.J. Integr. Plant Biol. 2013;55(4):294-388. DOI 10.1111/jipb.12041.Mähönen A.P., Bishopp A., Higuchi M., Nieminen K.M., Kinoshita K.,Törmäkangas K., Ikeda Y., Oka A., Kakimoto T., Helariutta Y. Cytokininsignaling and its inhibitor AHP6 regulate cell fate during vasculardevelopment. Science. 2006;311(5757):94-98. DOI 10.1126/science.1118875Mähönen A.P., Bonke M., Kauppinen L., Riikonen M., Benfey P.N.,Helariutta Y. A novel two-component hybrid molecule regulatesvascular morphogenesis of the Arabidopsis root. Genes Dev. 2000;14(23):2938-2943. DOI 10.1101/gad.189200Marhavý P., Bielach A., Abas L., Abuzeineh A., Duclercq J., Tanaka H.,Pařezová M., Petrášek J., Friml J., Kleine-Vehn J., Benková E. Cytokininmodulates endocytic trafficking of PIN1 auxin efflux carrierto control plant organogenesis. Dev. Cell. 2011;21(4):796-804. DOI10.1016/j.devcel.2011.08.014.Ménard D., Pesquet E. Cellular interactions during tracheary elementsformation and function. Curr. Opin. Plant Biol. 2015;23:109-115.DOI 10.1016/j.pbi.2014.12.001.Mironova V., Teale W., Shahriari M., Dawson J., Palme K. The systemsbiology of auxin in developing embryos. Trends Plant Sci. 2017;22(3):225-235. DOI 10.1016/j.tplants.2016.11.010.Miyashima S., Roszak P., Sevilem I., Toyokura K., Blob B., Heo J.O.,Mellor N., Help-Rinta-Rahko H., Otero S., Smet W., BoekschotenM., Hooiveld G., Hashimoto K., Smetana O., Siligato R.,Wallner E.S., Mähönen A.P., Kondo Y., Melnyk C.W., Greb T., NakajimaK., Sozzani R., Bishopp A., De Rybel B., Helariutta Y. MobilePEAR transcription factors integrate positional cues to primecambialgrowth. Nature. 2019;565(7740):490-494. DOI 10.1038/s41586-018-0839-y.Miyashima S., Sebastian J., Lee J.Y., Helariutta Y. Stem cell functionduring plant vascular development. EMBO J. 2013;32(2):178-193.DOI 10.1038/emboj.2012.301.Möller B.K., ten Hove C.A., Xiang D., Williams N., López L.G.,YoshidaS., Smit M., Datla R., Weijers D. Auxin response cell-autonomouslycontrols ground tissue initiation in the early Arabidopsisembryo. Proc. Natl. Acad. Sci. USA. 2017;114(12):2533-2539. DOI 10.1073/pnas.1616493114.Möller B., Weijers D. Auxin control of embryo patterning. Cold SpringHarb. Perspect. Biol. 2009;1(5):a001545. DOI 10.1101/cshperspect.a001545.Muraro D., Mellor N., Pound M.P., Help H., Lucas M., Chopard J.,ByrneH.M., Godin C., Hodgman T.C., King J.R., Pridmore T.P.,Helariutta Y., Bennett M.J., Bishopp A. Integration of hormonalsignaling networks and mobile microRNAs is required for vascularpatterning in Arabidopsis roots. Proc. Natl. Acad. Sci. USA. 2014;111(2):857-862. DOI 10.1073/pnas.1221766111.Nieminen K., Blomster T., Helariutta Y., Mähönen A.P. Vascularcambiumdevelopment. Arabidopsis Book. 2015;13:e0177. DOI10.1199/tab.0177.Ohashi-Ito K., Bergmann D.C. Regulation of the Arabidopsis root vascularinitial population by LONESOME HIGHWAY. Development.2007;134(16):2959-2968. DOI 10.1242/dev.006296.Ohashi-Ito K., Matsukawa M., Fukuda H. An atypical bHLH transcriptionfactor regulates early xylem development downstream of auxin.Plant Cell Physiol. 2013;54(3):398-405. DOI 10.1093/pcp/pct013.Ohashi-Ito K., Saegusa M., Iwamoto K., Oda Y., Katayama H., KojimaM., Sakakibara H., Fukuda H. A bHLH complex activates vascularcell division via cytokinin action in root apical meristem. Curr.Biol. 2014;24(17):2053-2058. DOI 10.1016/j.cub.2014.07.050.Palovaara J., Saiga S., Wendrich J.R., van’t Wout Hofland N., vanSchayck J.P., Hater F., Mutte S., Sjollema J., Boekschoten M.,HooiveldG.J., Weijers D. Transcriptome dynamics revealed bya gene expression atlas of the early Arabidopsis embryo. Nat.Plants. 2017;3(11):894-904. DOI 10.1038/s41477-017-0035-3.Qian P., Song W., Zaizen-Iida M., Kume S., Wang G., Zhang Y., Kinoshita-Tsujimura K., Chai J., Kakimoto T. A Dof-CLE circuit controlsphloem organization. Nat. Plants. 2022;8(7):817-827. DOI 10.1038/s41477-022-01176-0.Ren S.C., Song X.F., Chen W.Q., Lu R., Lucas W.J., Liu C.M.CLE25 peptide regulates phloem initiation in Arabidopsis througha CLERK-CLV2 receptor complex. J. Integr. Plant. Biol. 2019;61(10):1043-1061. DOI 10.1111/jipb.12846.Robert H.S., Crhak Khaitova L., Mroue S., Benková E. The importanceof localized auxin production for morphogenesis of reproductiveorgans and embryos in Arabidopsis. J. Exp. Bot. 2015;66(16):5029-5042. DOI 10.1093/jxb/erv256.Rodriguez-Villalon A. Wiring a plant: genetic networks for phloemformation in Arabidopsis thaliana roots. New Phytol. 2016;210(1):45-50. DOI 10.1111/nph.13527.Rodriguez-Villalon A., Gujas B., van Wijk R., Munnik T., Hardtke C.S.Primary root protophloem differentiation requires balanced phosphatidylinositol-4,5-biphosphate levels and systemically affects rootbranching. Development. 2015;142(8):1437-1446. DOI 10.1242/dev.118364.Ross-Elliott T.J., Jensen K.H., Haaning K.S., Wager B.M., KnoblauchJ., Howell A.H., Mullendore D.L., Monteith A.G., Paultre D.,Yan D., Otero S., Bourdon M., Sager R., Lee J.Y., Helariutta Y.,Knoblauch M., Oparka K.J. Phloem unloading in Arabidopsis rootsis convective and regulated by the phloem-pole pericycle. eLife.2017;6:e24125. DOI 10.7554/eLife.24125.Roszak P., Heo J.O., Blob B., Toyokura K., Sugiyama Y., de LuisBalaguer M.A., Lau W.W.Y., Hamey F., Cirrone J., Madej E., BouattaA.M., Wang X., Guichard M., Ursache R., Tavares H., Verstaen K.,Wendrich J., Melnyk C.W., Oda Y., Shasha D., Ahnert S.E., Saeys Y.,De Rybel B., Heidstra R., Scheres B., Grossmann G., Mähönen A.P.,Denninger P., Göttgens B., Sozzani R., Birnbaum K.D., Helariutta Y.Cell-by-cell dissection of phloem development links a maturationgradient to cell specialization. Science. 2021;374(6575):eaba5531.DOI 10.1126/science.aba5531Ruiz Sola M.A., Coiro M., Crivelli S., Zeeman S.C., Hansen S.S.K.,Truernit E. OCTOPUS-LIKE 2, a novel player in Arabidopsis rootand vascular development, reveals a key role for OCTOPUS familygenes in root metaphloem sieve tube differentiation. New Phytol.2017;216(4):1191-1204. DOI 10.1111/nph.14751.Růžička K., Ursache R., Hejátko J., Helariutta Y. Xylem development–from the cradle to the grave. New Phytol. 2015;207(3):519-535.DOI 10.1111/nph.13383.Sauer M., Balla J., Luschnig C., Wisniewska J., Reinöhl V., Friml J.,Benková E. Canalization of auxin flow by Aux/IAA-ARF-dependentfeedback regulation of PIN polarity. Genes Dev. 2006;20(20):2902-2911. DOI 10.1101/gad.390806.Scarpella E., Meijer A.H. Pattern formation in the vascular system ofmonocot and dicot plant species. New Phytol. 2004;164(2):209-242.DOI 10.1111/j.1469-8137.2004.01191.x.Scheres B., Wolkenfelt H., Willemsen V., Terlouw M., Lawson E.,Dean C., Weisbeek P. Embryonic origin of the Arabidopsis primaryroot and root meristem initials. Development. 1994;120(9):2475-2487. DOI 10.1242/dev.120.9.2475.Schlereth A., Möller B., Liu W., Kientz M., Flipse J., Rademacher E.H.,Schmid M., Jürgens G., Weijers D. MONOPTEROS controls embryonicroot initiation by regulating a mobile transcription factor.Nature. 2010;464(7290):913-916. DOI 10.1038/nature08836.Seo M., Kim H., Lee J.Y. Information on the move: vascular tissuedevelopment in space and time during postembryonic root growth.Curr. Opin. Plant Biol. 2020;57:110-117. DOI 10.1016/j.pbi.2020.08.002.Sjolund R.D. The phloem sieve element: a river runs through it. PlantCell. 1997;9(7):1137-1146. DOI 10.1105/tpc.9.7.1137.Smet W., Sevilem I., de Luis Balaguer M.A., Wybouw B., Mor E.,Miyashima S., Blob B., Roszak P., Jacobs T.B., Boekschoten M.,Hooiveld G., Sozzani R., Helariutta Y., De Rybel B. DOF2.1 controlscytokinin-dependent vascular cell proliferation downstream ofTMO5/LHW. Curr. Biol. 2019;29(3):520-529.e6. DOI 10.1016/j.cub.2018.12.041.Smetana O., Mäkilä R., Lyu M., Amiryousefi A., Sánchez Rodríguez F.,Wu M.-F., Solé-Gil A., Leal Gavarrón M., Siligato R., MiyashimaS., Roszak P., Blomster T., Reed J.W., Broholm S., Mähönen A.P.High levels of auxin signalling define the stem-cell organizer of thevascular cambium. Nature. 2019;565(7740):485-489. DOI 10.1038/s41586-018-0837-0.Smit M.E., Llavata-Peris C.I., Roosjen M., van Beijnum H., NovikovaD., Levitsky V., Sevilem I., Roszak P., Slane D., Jürgens G.,Mironova V., Brady S.M., Weijers D. Specification and regulationof vascular tissue identity in the Arabidopsis embryo. Development.2020;147(8):dev186130. DOI 10.1242/dev.186130.Smit M.E., Weijers D. The role of auxin signaling in early embryopattern formation. Curr. Opin. Plant Biol. 2015;28:99-105. DOI10.1016/j.pbi.2015.10.001.Smith R.A., Schuetz M., Roach M., Mansfield S.D., Ellis B., SamuelsL. Neighboring parenchyma cells contribute to Arabidopsisxylem lignification, while lignification of interfascicular fibers iscell autonomous. Plant Cell. 2013;25(10):3988-3999. DOI 10.1105/tpc.113.117176.Stadler R., Wright K.M., Lauterbach C., Amon G., Gahrtz M., FeuersteinA., Oparka K.J., Sauer N. Expression of GFP-fusions in Arabidopsiscompanion cells reveals non-specific protein traffickinginto sieve elements and identifies a novel post-phloem domain inroots. Plant J. 2005;41(2):319-331. DOI 10.1111/j.1365-313X.2004.02298.x.Tanaka H., Dhonukshe P., Brewer P.B., Friml J. Spatiotemporal asymmetricauxin distribution: a means to coordinate plant development.Cell. Mol. Life Sci. 2006;63(23):2738-2754. DOI 10.1007/s00018-006-6116-5.Truernit E. Sieve elements and their cell neighbours in the Arabidopsisroot – roles and relationships. J. Plant Physiol. 2022;268:153569.DOI 10.1016/j.jplph.2021.153569.Tvorogova V.E., Osipova M.A., Doduyeva I.E., Lutova L.A. Interactionbetween transcriptional factors and phytohormones in regulationof plant meristems activity. Ekologicheskaia Genetika = EcologicalGenetics. 2012;10(3):28-40. (in Russian)Vera-Sirera F., De Rybel B., Úrbez C., Kouklas E., Pesquera M.,Álvarez-Mahecha J.C., Minguet E.G., Tuominen H., Carbonell J.,Borst J.W., Weijers D., Blázquez M.A. A bHLH-based feedbackloop restricts vascular cell proliferation in plants. Dev. Cell. 2015;35(4):432-443. DOI 10.1016/j.devcel.2015.10.022.Verma S., Attuluri V.P.S., Robert H.S. An essential function for auxinin embryo development. Cold Spring Harb. Perspect. Biol. 2021;13(4):a039966. DOI 10.1101/cshperspect.a039966.Wabnik K., Robert H.S., Smith R.S., Friml J. Modeling framework forthe establishment of the apical-basal embryonic axis in plants. Curr.Biol. 2013;23(24):2513-2518. DOI 10.1016/j.cub.2013.10.038.Wallner E.S., López-Salmerón V., Belevich I., Poschet G., Jung I.,Grünwald K., Sevilem I., Jokitalo E., Hell R., Helariutta Y., Agustí J.,Lebovka I., Greb T. Strigolactone- and karrikin-independent SMXLproteins are central regulators of phloem formation. Curr. Biol.2017;27(8):1241-1247. DOI 10.1016/j.cub.2017.03.014.Weijers D., Jürgens G. Auxin and embryo axis formation: the ends insight? Curr. Opin. Plant Biol. 2005;8(1):32-37. DOI 10.1016/j.pbi.2004.11.001.Yang B., Minne M., Brunoni F., Plačková L., Petřík I., Sun Y., Nolf J.,Smet W., Verstaen K., Wendrich J.R., Eekhout T., Hoyerová K.,van Isterdael G., Haustraete J., Bishopp A., Farcot E., Novák O.,Saeys Y., de Rybel B. Non-cell autonomous and spatiotemporalsignalling from a tissue organizer orchestrates root vascular development.Nat. Plants. 2021;7(11):1485-1494. DOI 10.1038/s41477-021-01017-6.


Several antibiotics active against carbapenemase-producing organisms have recently been licensed for clinical use. These organisms usually have multiple integrative and conjugative elements with different resistance genes. The positioning of these agents for empirical use in sepsis caused by different carbapenemase-producing organisms is important in optimizing the likelihood of appropriate treatment before susceptibility testing is complete. Here we present the activities of meropenem-vaborbactam, imipenem-relebactam, eravacycline and tigecycline against NDM- and OXA-48-producing clinical isolates in a London teaching hospital.Klebsiella pneumoniae (17), Escherichia coli (10), Enterobacter aerogenes (3) and Serratia marcescens (1). NDM producers comprised K. pneumoniae (11), E. coli (10), Acinetobacter baumannii (3), Klebsiella oxytoca (1), Enterobacter asburiae (1), Enterobacter cloacae (1), Pseudomonas aeruginosa (1), Citrobacter koseri (1), Citrobacter freundii (1) and Leclercia adecarboxylata (1). MICs of meropenem/vaborbactam, imipenem/relebactam, eravacycline and tigecycline were determined using Etests. Interpretation was done using EUCAST breakpoints.Sixty-two previously characterized carbapenemase-producing Gram-negative clinical isolates were studied. These comprised 31 NDM and 31 OXA-48 producers. OXA-48 producers comprised OXA-48 producers: Eight of 17 K. pneumoniae, 8/10 E. coli and 2/3 E. aerogenes isolates were meropenem/vaborbactam susceptible. Of these, 2 K. pneumoniae, 5 E. coli and 1 E. aerogenes isolates were imipenem/relebactam susceptible. All imipenem/relebactam/susceptible isolates were also meropenem/vaborbactam susceptible. The S. marcescens isolate (1/1) was resistant to both antibiotics. The only isolates demonstrating susceptibility to the glycylcyclines were E. coli. Five of 10 were susceptible to both glycylcyclines. One of 10 was susceptible to one glycylcycline but resistant to the other. NDM producers: Five of 11 K. pneumoniae, 6/10 E. coli and 1/1 K. oxytoca, E. asburiae and C. koseri isolates were susceptible to meropenem/vaborbactam. Of these, two K. pneumoniae and the K. oxytoca (1/1) and E. asburiae (1/1) isolates were imipenem/relebactam susceptible. The L. adecarboxylata isolate (1/1) was meropenem/vaborbactam resistant but imipenem/relebactam susceptible. All P. aeruginosa, C. freundii, A. baumannii and E. cloacae isolates were resistant to both meropenem/vaborbactam and imipenem/relebactam. Seven of 10 E. coli isolates were tigecycline susceptible of which 6 were also eravacycline susceptible. One of 11 K. pneumoniae, 1/1 K. oxytoca and 1/1 L. adecarboxylata isolates were susceptible to both glycylcyclines. All other isolates were resistant to both glycylcyclines.E. coli than K. pneumoniae. Meropenem/vaborbactam provided better cover than imipenem/relebactam. Eravacycline and tigecycline showed similar activity to each other, but their activity was inferior compared with the β-lactam combinations, particularly against K. pneumoniae.In both OXA-48 and NDM producers, the activity of the four antibiotics tested was better against


Hcrt), tachykinin receptor 3 (Tacr3), cocaine and amphetamine-regulated transcript (Cart) and catecholamine-related biological processes; dopa decarboxylase (Ddc), histidine decarboxylase (Hdc), tyrosine hydroxylase (Th), and vasoactive intestinal peptide (Vip). In BACHD mice, few hypothalamic genes were differentially expressed compared to age-matched WT controls. However, GSEA indicated an enrichment of inflammatory- and gonadotropin-related processes at 10 months. In conclusion, we show that both wtHTT and mHTT overexpression change hypothalamic transcriptome profile, specifically mHTT, altering neuroendocrine circuits. In contrast, the ubiquitous expression of full-length mHTT in the BACHD hypothalamus moderately affects the transcriptomic profile.Structural changes and neuropathology in the hypothalamus have been suggested to contribute to the non-motor manifestations of Huntington’s disease (HD), a neurodegenerative disorder caused by an expanded cytosine-adenine-guanine (CAG) repeat in the huntingtin (HTT) gene. In this study, we investigated whether hypothalamic HTT expression causes transcriptional changes. Hypothalamic RNA was isolated from two different HD mouse models and their littermate controls; BACHD mice with ubiquitous expression of full-length mutant HTT (mHTT) and wild-type mice with targeted hypothalamic overexpression of either wild-type HTT (wtHTT) or mHTT fragments. The mHTT and wtHTT groups showed the highest number of differentially expressed genes compared to the BACHD mouse model. Gene Set Enrichment Analysis (GSEA) with leading-edge analysis showed that suppressed sterol- and cholesterol metabolism were shared between hypothalamic wtHTT and mHTT overexpression. Most distinctive for mHTT overexpression was the suppression of neuroendocrine networks, in which qRT-PCR validation confirmed significant downregulation of neuropeptides with roles in feeding behavior; hypocretin neuropeptide precursor ( HTT) gene  is a fatal neurodegenerative disorder caused by a CAG repeat expansion in exon one in the huntingtin (TT) gene . The expTT) gene . The lenTT) gene . Current alleles , which sThe hypothalamus plays a primary role in the central-peripheral regulatory network that maintains body homeostasis, including regulating whole-body energy metabolism . MetabolTranscriptional dysregulation in the striatum is one of the hallmarks of HD . SpecifiAll the mice used in the study were housed in groups and maintained at a 12 h light/dark cycle with free access to a standard chow diet and water. All the experimental procedures performed on mice were carried out in accordance with the approved guidelines in the ethical permits approved by Lund University Animal Welfare and Ethics committee in the Lund-Malmö region .Microarray profiling of the hypothalamic transcriptome was performed in Adeno-associated viral (AAV) vector-mediated groups of WT mice with targeted expression of wtHTT (AAV-HTT853-18Q) or mHTT (AAV-HTT853-79Q) fragments and BACHD mice that express full-length mHTT (97Q) . Both HDn = 5, 79Q: n = 8 and WT control: n = 5.AAV vector-mediated HD models achieve region-specific overexpression of HTT fragments in the brain through targeted injections using stereotactic surgery. AAV groups were assessed at 4 weeks post-injection since this was the earliest timepoint for a significant weight gain, as shown in previous studies . The vecn = 6 and WT: n = 6, and for 10 months of age: BACHD: n = 5 and WT: n = 3.Bacterial artificial chromosome-mediated transgenic mouse model is a transgenic mouse model of HD and ubiquitously expresses a full-length human mHTT  . In the Hypothalamic tissue was dissected on ice and snap-frozen in liquid nitrogen after a terminal dose of sodium pentobarbital  via intraperitoneal injection. Total RNA was extracted using the RNeasy Lipid Tissue Mini Kit  according to the manufacturer’s instructions. RNA concentration and RNA quality measured in terms of RNA integrity number (RIN) were determined using the Agilent 2,100 Bioanalyzer . Samples with poor RIN (<7) were omitted from further analysis. Microarray analysis was performed on total hypothalamic RNA using the Affymetrix platform .Analyses were made using R v.4.1.1 . Raw .CEp-value followed by sorting it from the highest log2 (FC) to the lowest. The top 10 most upregulated and top 10 most downregulated significantly differentially expressed genes were identified, and their respective RMA-limma values were used to construct and color the heatmap. Heatmaps were generated using the pheatmap package in R1 (v. 1.0.12) (Limma output files for 18Q vs. WT and 79Q vs. WT were sorted based on adj.  1.0.12) . Hierarcp-value < 0.05. The 18Q vs. WT and 79Q vs. WT datasets were subsequently sorted into three gene sets: shared genes, unique genes for 18Q vs. WT, and unique genes for 79Q vs. WT. The shared and unique gene lists (ENTREZ IDs) were imported into DAVID2 . The reported p-values were further adjusted using the Benjamini-Hochberg procedure to correct for multiple testing. Outputs from GSEA can be found in ClusterProfiler (v.4.0.5) was used to perform GSEA . The funTo synthesize cDNA, 1 μg of RNA from each sample was reverse transcribed using SuperScript IV Reverse Transcriptase SuperScript IV kit  according to the manufacturer’s instructions. Mouse qRT-PCR primers were designed using Primer3Plus software . qRT-PCRp-value < 0.05 considered statistically significant.Statistical analysis of qRT-PCR data was performed using Graphpad Prism 9 . . Data were analyzed using non-parametric Mann-Whitney U tests with a p-value in the limma datasets comparing injected mice to uninjected (18Q vs. WT and 79Q vs. WT) showed a skewed distribution with a preference for upregulated genes in the injected mice  of the microarray data showed a clear separation of the AAV-HTT groups from the WT samples . PCA of ted mice . Filteri5 cutoff .p < 0.05 cutoff, 1,422 variables remained for BACHD 2 months vs. WT  variables, and we further analyzed the data using the Database for Annotation, Visualization and Integrated Discovery (DAVID) (see text footnote 2) (p < 0.05) identified by limma between the 18Q vs. WT and 79Q vs. WT datasets (The 18Q vs. WT and 79Q vs. WT datasets had the highest number of significant (adj. tnote 2) . Previoutnote 2) . Furthertnote 2) . Therefodatasets , using tdatasets . DAVID Fn = 51 genes, (adj. p < 0.05) to 79Q vs. WT (adj. p > 0.05)] showed that the gene with the highest difference in log2(FC) was immunoglobulin heavy chain (X24 family) (Igh-VX24) followed by tripartite motif-containing 30D (Trim30d) and toll-like-receptor 7 (Tlr7) . Across all 51 genes, the mean difference with 95% CI was 0.135   > 0 and 61 with log2(FC) < 0 . Functio, 0.181) , 2.n = 12 genes, adj. p < 0.05) to 18Q vs. WT (adj. p > 0.05) showed a mean difference with 95% CI of -0.042 . In the top 5/10 cluster related to neuroactive ligands , we found a mean difference in log2(FC) of -0.067  between the 79Q vs. WT and 18Q vs. WT datasets, where the genes with the highest difference were proenkephalin (Penk) and tachykinin receptor 3 (Tacr3)   > 0 and 131 with log2(FC) < 0 . Among t: -0.24] , 2.Ldlr; log2(FC) 18Q vs. WT: −0.49, 79Q vs. WT: −0.54] (The shared gene list between 18Q vs. WT and 79Q vs. WT consisted of 410 genes with log2(FC) > 0 and 39 genes with log2(FC) < 0 . An immu: −0.54] , 2.p and log2(FC)] in the 18Q vs. WT and 79Q vs. WT datasets , and major facilitator superfamily domain-containing 2A (Mfsd2a) (Nms), Hdc, synaptic vesicle glycoprotein 2C (Sv2c), and Tacr3  , genes tnd Tacr3 . CompariNext, we elaborated further on the functional implications of the differences in the top 10 downregulated genes between 18Q vs. WT and 79Q vs. WT. GSEA of GO-BP with leading-edge analysis  was usedp-value < 0.05 for the 79Q vs. 18Q and BACHD datasets. By using GSEA, we may still identify biologically relevant pathways that exhibit noteworthy cross-correlation between genes with a subtle change in expression levels or weak statistical significance  was -0.156 . The leading- edge gene with the lowest log2(FC) was prolactin (Prl) at -1.63 followed by growth hormone (Gh) -1.02, Vip -0.59, the alpha subunit of glycoprotein hormones (Cga) -0.53 and hypocretin (Hcrt) -0.35  -0.35 , 4.Acot5) and vesicular glutamate transporter 1 (Slc17a7) that were respectively part of the fatty-acid related and “Glutamatergic synapse” pathways . None of the three pathways identified in 2 months old BACHD vs. WT were significant in GSEA-KEGG for 10 months old BACHD vs. WT. When comparing each leading-edge gene set in 2 months old BACHD vs. WT to the same genes in 10 months BACHD vs. WT, the mean log2(FC) difference was <0.2 for all three pathways. Instead, for 10 months old BACHD vs. WT, fourteen KEGG pathways were identified (H2-Aa), and Cd74 were among the leading-edge genes with the highest log2(FC) and difference from the 2 months of age vs. WT dataset . Pathways related to gonadotropin-releasing hormone (Gnrh), all NES > 0, were also found in 10 months old BACHD vs. WT, where the alpha subunit of glycoprotein hormones (Cga) was among the leading-edge genes with the highest fold change . Only one of the 14 KEGG pathways identified by GSEA had a negative NES of -1.95, indicating suppression; “Oxidative phosphorylation” (mmu00190). The overall change of the 59 genes in the leading-edge gene set was -0.056  [mean log2(FC) with 95% confidence interval (95% CI)].For 2 months old BACHD vs. WT, GSEA-KEGG identified three pathways; “Glutamatergic synapse” , “Biosynthesis of unsaturated fatty acids”  and “Fatty acid elongation”  . Among lentified . Immune-entified . HistocoHcrt) in mice with mHTT 79Q overexpression compared to wtHTT 18Q groups and uninjected WT controls  that was among the top suppressed processes in GSEA-GO BP consisted of 57 genes . We havecontrols . Here, imparison .Hdc as part of the top 10 downregulated genes and related leading-edge subsets in GSEA. Comparing GSEA-GO BP outputs between the 79Q vs. WT and 18Q vs. WT datasets showed a higher number of catecholamine-related processes that were only significant for the 79Q vs. WT dataset  or Growth hormone-releasing hormone (Ghrh)  . A matchransport .Cart, Tacr3, Hcrt, Vip, Th, and Ghrh, only Tacr3 was significantly downregulated compared to the age-matched WT controls.Next, we performed qRT-PCR of a subset of genes analyzed for the HTT-AAV groups in the BACHD 2 months group . Among CHdc mRNA have been reported in HD patients . Among leading-edge genes was Cga, which encodes the alpha subunit of glycoprotein hormones   . Furthereriphery , notableeriphery . Expresseriphery . Furthereriphery . Similareriphery . In lineeriphery . As HD periphery . TherefoStoml3 and Sv2c associated with dopaminergic signal transmission were among the top 10 differentially downregulated in 79Q vs. WT .RS-K, ÅP, ED, and MB designed the experiments from RS-K and ÅP. ED, AD, RS-K, and SL performed the experiments. ED, NA, AD, and RS-K analyzed the data. ED, RS-K, and AD wrote the first draft of the manuscript. All authors reviewed the manuscript and approved the final version.


TM) can ameliorate liver fibrogenesis induced by thioacetamide (TAA) treatment. Our results showed that the TAA treatment caused lower body weight gains and enlarged livers, as well as higher serum ALT, AST, and ALP levels (p < 0.05). This liver inflammatory and fibrotic evidence was ameliorated (p < 0.05) by supplementing with GBHP01TM; this partially resulted from its antioxidant abilities, including decreased TBARS values but increased TEAC levels, reduced GSH contents and catalase/GPx activities in the livers of TAA-treated rats (p < 0.05). Additionally, fewer nodules were observed in the appearance of the livers of TAA-treated rats after supplementing with GBHP01TM. Similarly, supplementing GBHP01TM decreased fibrotic scars and the fibrotic score in the livers of TAA-treated rats (p < 0.05). Moreover, the increased hepatic IL-6, IL-1β, and TNF-α levels after TAA treatment were also alleviated by supplementing with GBHP01TM (p < 0.05). Meanwhile, GBHP01TM could decrease the ratio of LC3B II/LC3B I, but upregulated P62 and Rab7 in the livers of TAA-treated rats (p < 0.05). Taking these results together, the CLH-based supplement (GBHP01TM) can be characterized as a natural agent against liver fibrogenesis.Chicken-liver hydrolysates (CLHs) have been characterized as performing several biofunctions by our team. This study aimed to investigate if a CLH-based supplement (GBHP01 Liver cancer ranks third among cancer categories globally, and approximately 830,000 people die with liver cancer every year . SimilarFood-derived protein hydrolysates have been suggested to provide functional properties such as antioxidant , lipid-lCandidates for functional-food ingredients should be either effective or low in cost. Regarding the poultry industry in Taiwan, approximately 10,000 metric tons of chicken livers (2.5% based on broilers’ wt.) have been produced in recent years  and antiTM) containing 200 mg CLH powder and 20 mg hesperidin  per capsule was generously offered by Great Billion Biotech Co., Ltd., New Taipei City, Taiwan. The composition of free amino acids and imidazole-ring dipeptides in a GBHP01TM capsule were sent for analysis using an Amino Acid Analyzer  in the Food Industry Research and Development Institute . The total contents of free essential and non-essential amino acids and imidazole-ring dipeptides are 13.10, 65.04, and 0.07 mg per capsule (average 650 mg), respectively, where the total free branched-chain amino acid (BCAA) and taurine content are 5.77 mg and 51.67 mg per capsule ; (2) TAA: 100 mg TAA/kg BW (i.p.) + 1.5 mL pure H2O ; (3) TAA + SIL: 100 mg TAA/kg BW (i.p.) + 150 mg silymarin/kg BW in 1.5 mL pure H2O ; (4) TAA + GBHP01(1X): 100 mg TAA/kg BW (i.p.) + 133 mg GBHP01TM content/kg BW in 1.5 mL pure H2O ; (5) TAA + GBHP01(2X): 100 mg TAA/kg BW (i.p.) + 266 mg GBHP01TM content/kg BW in 1.5 mL pure H2O . It was reported that silymarin could ameliorate livre fibrogenesis via decreasing endoplasmic reticulum stress-related gene expressions, inflammatory cytokines, collagen accumulation, and matrix metalloproteinase (MMP-2 and MMP-9) activities, as well as increasing tissue inhibitors of metalloproteinase gene expressions (TIMP-1 and TIMP-3) in livers . Additionally, serum ALB values were reduced (p < 0.05) by TAA treatment, except in the TAA + GBHP01(2X) group. Focusing on serum lipid levels, no (p > 0.05) differences on TC levels were observed among groups. The TAA group had the lowest (p < 0.05) serum TG levels among groups, but a reverse (p < 0.05) effect was observed in silymarin and GBHP01TM-supplemented groups, while TAA + SIL and TAA + GBHP01(2X) groups even showed similar (p > 0.05) serum TG levels to the CON group.After 8 weeks of experiment, TAA treatment resulted in the lower body weight of rats (approximately a 12.19% reduction), while lower weight increases and smaller food intakes were observed in TAA-treated rats than in control rats ( < 0.05) . No , while the silymarin or GBHP01TM-supplemented group had similar (p > 0.05) TBARS to the CON group and no (p > 0.05) difference in TEAC levels among GBHP01TM-supplemented groups and the CON group. Besides, the decreased pattern of reduced GSH levels was observed in TAA-treated groups, but silymarin or GBHP01TM supplementation allowed for maintenance of the reduced GSH level in TAA-treated rats, especially in the group receiving 2X doses of GBHP01TM supplementation (p < 0.05). With regard to antioxidant enzymatic activities, SOD activities were not (p > 0.05) different among groups. The TAA group showed reduced (p < 0.05) CAT and GPx activities compared to CON group, while silymarin or GBHP01TM supplementation avoided reducing CAT activities, showing a result similar (p > 0.05) even to that of the CON group; reversed (p < 0.05) effects on GPx activities were only observed in GBHP01TM supplemented groups. In hepatic proinflammatory cytokines, the TAA group had higher (p < 0.05) hepatic IL-1β , IL-6 , and TNF-α (28.89% increase) than the CON group. GBHP01TM supplementation reduced (p < 0.05) these increased hepatic inflammatory cytokines of TAA-treated rats to levels similar (p > 0.05) even to those of the CON group, but silymarin supplementation only reduced the hepatic IL-6 level.As the results in TM supplementation. Via H&E stained observation  according to the H&E stains were increased by TAA treatments, while significant reductions (p < 0.05) in all three HAI scores were observed in the GBHP01TM supplementation group; however, silymarin only demonstrated a reduced pattern  score. Silymarin treatment did not (p > 0.05) attenuate the liver Metavir score in the TAA-treated rats. Treatment with 1X or 2X GBHP01 supplement decreased (p < 0.05) the scores in TAA-treated rats, although the scores were still higher (p < 0.5) than those of the CON group , but GBHP01TM supplement significantly downregulated (p < 0.05) this ratio, reaching levels even similar (p > 0.05) to those of CON (p < 0.05) by TAA treatment (TM supplementation significantly upregulated (p < 0.05) these two proteins in livers of TAA-treated rats; meanwhile, the P62 and Rab7 protein expression in the livers of the TAA + GBHP01(1X) and TAA + GBHP01(2X) groups were reversed to similar levels to those of the CON group (p > 0.05).With regard to autophagy modulation in livers of TAA-treated rats, the ratio of LC3B II/LC3B I of TAA group was almost 1.8 relative fold to that of CON group  and hesperidin, which are both characterized by antioxidant abilities , only 4.1 and 8.2 mg hesperidin/kg BW were given to rats. Hence, it was assumed that the amelioration of TAA-induced liver fibrosis by hesperidin can be considered negligible in this study. To summarize, this regulation probably mainly results from the bioactive ingredients in the CLHs of GBHP01TM supplements  . Accordin (IL10) , the revsurvival . Recentlsurvival  and incrsurvival . It was survival . It was survival . Besidessurvival . This phsurvival ,37. LC3Bsurvival , while Rsurvival . As the survival . Meanwhisurvival . Taurinesurvival . Similarsurvival . As explturation . Althougin/kg BW ,43,44. BTM) showed an ameliorating effect against liver fibrogenesis induced by TAA treatment  against liver fibrogenesis may be attributed to the bioactive ingredients in CLHs, including anserine, taurine, glutamic acid, aspartic acid, BCAAs, etc.In this study, chicken liver hydrolysate (CLH)-based supplements (GBHP01reatment . The hep