Earth’s geothermal flux drives vigorous flows that can suspend microorganisms and sediment in isolated Antarctic subglacial lakes. Trapped beneath the Antarctic ice sheet lie over 400 subglacial lakes, which are considered to be extreme, isolated, yet viable habitats for microbial life. The physical conditions within subglacial lakes are critical to evaluating how and where life may best exist. Here, we propose that Earth’s geothermal flux provides efficient stirring of Antarctic subglacial lake water. We demonstrate that most lakes are in a regime of vigorous turbulent vertical convection, enabling suspension of spherical particulates with diameters up to 36 micrometers. Thus, dynamic conditions support efficient mixing of nutrient- and oxygen-enriched meltwater derived from the overlying ice, which is essential for biome support within the water column. We caution that accreted ice analysis cannot always be used as a proxy for water sampling of lakes beneath a thin (<3.166 kilometers) ice cover, because a stable layer isolates the well-mixed bulk water from the ice-water interface where freezing may occur. The Antarctic continent is covered with ice, growing and shrinking over periods of tens to hundreds of thousands of years, since at least the last 14 million years  the average geothermal flux, for a wide range of geophysical conditions and conclude that most Antarctic subglacial lakes are unstable to convection. We then demonstrate that most subglacial lakes . Convection in subglacial lakes is complex, because the thermal expansion coefficient of freshwater α, which indicates how fluid parcels contract or expand with changes in temperature , depends on water pressure p > pi and the temperature T itself  and increases with pressure and temperature, such that convection becomes more vigorous as F, h, and H increase. For ice covers less than the critical ice depth H*, or ice pressure pi < p* = 2848 dbar , however, the thermal expansion coefficient changes sign with temperature, such that density does not simply decrease with temperature but becomes a nonlinear and nonmonotonic function of T. Specifically, as is shown in H < H*, α increases with temperature but is first negative for Tf(pi) ≤ T < Td(p) (close to the ice ceiling), where Tf is the freezing temperature and Td is the temperature of maximum density, before becoming positive at higher temperature. Having α < 0 close to the ice ceiling for H < H* means that the density stratification is always stable at the top of the lake and that the bottom layer is buoyant only if the bottom temperature exceeds Td, i.e., such that the density stratification is top heavy near the bottom. Having α > 0 on the bottom boundary is a necessary condition for deep water masses to be buoyant but, however, is not sufficient for convection to set in. The geothermal flux must be also larger than the adiabatic heat flux and adequate to sustain a buoyancy force that can overcome viscous dissipation and thermal diffusion.Subglacial lakes can experience dynamic vertical convection flows [also known as Rayleigh-Bénard (RB) convection  toolbox  but then decreases with h and pi in most of the parameter space. We find Fc < 50 mW/m2 (as shown by the black isocontour labeled “50”), which is a typical background value for Earth’s geothermal flux around Antarctica, in most of the parameter space. We predict that Lake CECs and South Pole Lake (SPL) are unstable to vertical convection if subject to a 50 mW/m2 flux, i.e., their critical heat flux Fc is less than 50 mW/m2, despite having relatively thin ice covers H < H* (shown by the gray dashed line). Here, we have centered the vertical axis of p* by using the shifted ice pressure variable pi − p* and a symmetric logarithmic scale. As a result, the transition from a fully convective water column (for pi > p*) to a convective water column with a stably stratified upper layer (for pi ≤ p*) is smooth even though Fc increases rapidly as pi decreases below p*. H*. Thus, the pi = p* isobar, which separates lakes that are fully convective from lakes that are only partially convecting, is important not only for the theoretical calculation of Fc but also in practice. Almost half of the subglacial lakes (with pi < p*) may be expected to have a top layer that is stable, although possibly modified by the dynamics near the ice ceiling and overshooting convection. We remark that Fc is constrained primarily by the condition of having α > 0 on the bottom boundary for small pi and h (top left corner) and by the condition that it must exceeds the adiabatic flux for large pi and h (bottom right corner). In between, viscous dissipation and thermal diffusion dominate the calculation of Fc. Note that our prediction of Fc for small pi and large h is conservative and may overestimate the true Fc. We provide further details about the calculation of Fc in Materials and Methods and in the Supplementary Materials.F greater than the critical heat flux Fc, it is of interest to know whether the convective instability results in high or low flow velocities. In general, estimates of flow velocities require dedicated simulations or laboratory experiments. For the case of turbulent vertical convection, however, various scientific communities have proposed predictive laws of hydrodynamic variables based on control parameters, which can be used as leading-order estimates for turbulence intensity in convective subglacial lakes [see . The subscript F of FRa means that the Rayleigh number of subglacial lakes is a flux-based Rayleigh number, since it is based on a prescribed geothermal flux rather than a prescribed temperature difference, which is more common in idealized studies of natural convection , the characteristic turbulent flow velocity U, and the length scale of turbulence 𝓁, which represents the typical distance between thermal plumes , and a turbulent transition layer (just above the convective bulk), which is typically on the order of a few centimeters or smaller (thickness δt), i.e., δ = δS + δt. For ice thickness, H < 2000 m, δS is between 10 and 40 m. The stable layer thickness δS decreases with H and vanishes for H > H* (since α > 0 everywhere in this case), such that the full conductive layer is limited—for a thick ice cover—to a turbulent boundary layer attached to the ice ceiling, which is small. In all cases, the top stable layer transfers heat by conduction only. Thus, the temperature increases linearly from Tf(pi) near the ice to Tf(pi) + δF/k at the bottom of the stratified layer. The anomalous temperature of the well-mixed convective bulk (above freezing) can then be approximated as Tbulk = δF/k  and use in FRa, decreases rapidly across the p* isobar for small water depths. For instance, αeff decreases from 3 × 10−6 ° C−1 to 3 × 10−7 ° C−1 between pi = p* + 100 dbar and pi = p* for h = 10 m. We expect that the discontinuity would become less sharp but would not completely disappear upon relaxing our conservative approximation for αeff, because α will always be  much smaller in lakes with a thin ice cover (pi ≤ p*) than in lakes with a thick ice cover (pi > p*). We also show in Ulsc based on the scaling laws derived in (H = 3945 m (filled squares), which is the ice thickness above Lake Vostok. Ulsc is smaller than U (shown by the filled circles) by up to a factor 5 because the GL theory focuses on the velocity of the large-scale circulation, while U is the characteristic root-mean-square velocity of turbulent plumes  or one order of magnitude smaller  than the large-scale velocity of vertical convection (Ulsc).2 for a broad range of ice overburden pressures and water depths . Also, estimates of Fc exceeding 50 mW/m2 (as is the case for a lake 20 m deep and under 1 km of ice), hence suggesting stable subglacial lakes, could be verified qualitatively through direct sampling and revised if measurements demonstrate a dynamic environment.We have demonstrated that the critical heat flux leading to vertical convection in subglacial lakes is much less than 50 mW/mr depths . Thus, iH < H*). Here, we have used state-of-the-art scaling laws of RB convection and took into account the variability of the thermal expansion coefficient to make predictions about the thickness of the stable layer near the ice ceiling (δ), the anomalous temperature of the well-mixed turbulent bulk (Tbulk), the characteristic velocity of plumes (U), the characteristic velocity of the large-scale circulation (Ulsc), and the characteristic distance between plumes (𝓁). For completeness, we have also used state-of-the-art scaling laws of horizontal convection to make predictions about the typical horizontal velocity (Vhc) of the baroclinic flow that develops along a tilted ice-water interface.Vertical convection in subglacial lakes is different from vertical convection in the canonical RB problem, mainly because the thermal expansion coefficient (α) of freshwater in subglacial lakes changes with pressure and temperature , while iU ≈ 4 mm/s and Ulsc ≈ 1 mm/s for a 1-km deep lake. These vertical velocities are larger than the horizontal velocity associated with the baroclinic flow along a tilted ice-water interface, even if the ice slope is as large as s = 10−2. The ratio Ulsc/Vhc is not much larger than 1 for steep slopes. However, if we assume that the vertical velocity of the baroclinic flow scales like ∼Vhch/L with L ≫ h the horizontal length of the lake, then UlscL/(Vhch) ≫ 1. Thus, geothermal heating is a key factor—if not the dominant one—controlling hydrological conditions in Antarctic subglacial lakes. We provide predictions of flow properties for five well-studied subglacial lakes in The predictions for the different hydrodynamic variables are shown in Our analysis assumes that vertical convection and horizontal convection are decoupled. This limitation comes from the fact that numerical simulations and laboratory experiments tackling both dynamics simultaneously  are lacking. A handful of coarse numerical models have provided some insights into the large-scale circulation of select subglacial lakes  their density ρs is larger but of the same order as the density of water, i.e., ρs ≈ 3ρ0 with ρ0 ≈ 999 kg/m3 the mean density of water; and (iii) particulates’ loading is expected to be dilute (W = 2gr2(ρs − ρ0)/9η, assuming a linear Stokes drag, with η = 0.0017 m2 s−1 the dynamic viscosity of water. In a convecting fluid, particulates can either settle with a similar velocity or stay suspended provided that the large-scale circulation is upward and has velocity Ulsc > W, where, here, Ulsc is estimated from the GL theory. We report in the last column of rmax of particulates maintained in suspension by the large-scale flow, i.e., such that W(rmax) = Ulsc. We find 2rmax > 7.8 μm in all cases , which means that a broad range of particulates as observed in Vostok’s accreted ice, and qualifying as “fine silt,” may be suspended in all five lakes. For Lake Vostok, it may be noted that we predict a maximum radius (36 μm) larger than that (23 μm) reported by  ∼ ezW/D− in the bulk, which shows that increased turbulence increases the particulates’ concentration by raising the background effective diffusivity, which we denote by D, in the water column. The background effective diffusivity in the open ocean is well documented and typically ranges from D = 10−5 m2 s−1 to D = 10−4 m2 s−1  and decays by at least one order of magnitude every 10 m. This means that future explorations limited to sampling in the bulk of the lake would have to rely on intermittent plumes and local upwelling of the large-scale circulation to bring particulates upward. The mean number N of particulates in the water column, or turbidity, is key to fully assessing the habitability of subglacial lakes, in addition to the concentration of oxygen molecules derived from the ice above , turbulent length scales (1 m), top stable layer thickness (0.01 to 10 m), temperature fluctuations (0.001 to 1 K), and the radius of particulates suspended in the water column (1 to 40 μm) due to vertical convection in Antarctic subglacial lakes. Those predictions will be verifiable by future explorations sampling lake waters and sediments using, e.g., conductivity, temperature, and depth (CTD) profilers, such as envisioned for Lake CECs and as was initially planned for Lake Ellsworth  (We remark that having a stable density stratification at the top of the water column does not imply a completely quiescent environment adjacent to the ice ceiling (even if flat). Internal gravity waves , as a function of in situ temperature T, water pressure p, and absolute salinity S; (ii) the freezing temperature, pi (the pressure at the ice-water interface) and S; and (iii) the temperature of maximum density, p and S. Here, we restrict our attention to freshwater as opposed to seawater, i.e., we set S = 0, such that variables do not depend on S. We use superscript e to denote exact quantities, and we call the water pressure p the pressure for short, which is the absolute pressure minus atmospheric pressure pa = 10.1325 dbar. Note that p is the full water pressure, which includes pressure contributions from the ice cover, such that p > pi, with pi the ice overburden pressure. The ice pressure is related to the ice thickness through pi = ρigH/104, with pi in decibars and H in meters, and we assume a mean ice density ρi = 917 kg/m3. Unless stated otherwise, all variables use SI units except temperature variables, which are in degrees Celsius (°C) and pressure variables, which are in decibars (dbar), since °C and dbar are standard units in physical oceanography.We use the TEOS-10 toolbox in MATLAB T = Td(p). For 0 < p < 104 dbar and Tf < T < Tf + 15 K, we find that the best-fit bivariate polynomial (with p in dbar)e to within less than 0.01% relative error. This implies density errors less than 0.1 kg/m3, which is an order of magnitude less than density variations expected with temperature alone. From T = Td > Tf for pressures lower than p* = 2848 dbar.For simplicity, we derive explicit, approximate expressions for ρ, S ≈ 35 g/kg, the density of Earth’s oceans decreases monotonically with increasing temperatures.We note that the nonmonotonic, anomalous behavior of water density at low pressure is well known for freshwater lakes at atmospheric pressure  centred on a lake’s top boundary, the equations for the velocity vector u, pressure p, density ρ, and temperature T (in °C) read  in the above equations but is converted to dbar by dividing by 104 when used in The evolution equations for subglacial lakes are the Navier-Stokes equations in the Boussinesq approximation. Here, we include compressibility effects in the energy equation because we are interested in the calculation of the  critical heat flux at the onset of convection, but compressibility effects can otherwise be neglected when considering the (large) geothermal heat flux. In a Cartesian coordinates system  at z = 0 m, while at the base of the lake, it is the heat flux that is enforced, i.e., zTk∂ = − F at z = − h, with F > 0 the  heat flux and h > 0 the lake depth; also, p = pi at z = 0. 0 in the hydrostatic base-state equation, such that Subglacial lake water must be at the freezing temperature at the upper lake boundary, i.e., p = pi − ρ0gz (overbar dropped) to be locally  stable is , and we approximate ρ ≈ ρ0 at leading order. dTad/dz is known as the adiabatic temperature gradient and readsdTad/dz > 0 if α < 0. Here, heating is provided from the bottom of the lake such that we always have dT/dz < 0. As a result, when α < 0, equation dT/dz < dTad/dz < 0, i.e., if the heat flux exceeds  the adiabatic heat flux. Thus, the two conditions for subglacial lakes experiencing geothermal heating  to be locally unstable areThe criterion for an ideal (dissipationless) compressible fluid with hydrostatic base-state pressure dTaddz)<0where s idTaddz)<0where s idTad/dz are functions of z when considering the base state of a subglacial lake heated from below. Specifically, α increases with depth, while dTad/dz decreases with depth , such that equation Note that both α and Equations X) as1, Td, and C, e.g., Td1 = − 2.0059 × 10−3 K/dbar  as a function of wave number F for a range of input parameters . The critical minimum heat flux Fc that destabilizes the base state is the minimum of F for which there exists a wave number k⊥ such that σ > 0. We report Fc in h, such that we limit the calculations to h ≤ 20 m. We extrapolate to larger h using scaling laws that are either asymptotically valid or conservative, i.e., such that they may overestimate Fc. We describe the extrapolation procedure in detail in the Supplementary Materials.Since the perturbation equations do not depend explicitly on Tbulk, U, and 𝓁 for subglacial lakes subject to a geothermal flux F = 50 mW/m2.We show in the Supplementary Materials that vertical convection in Antarctic subglacial lakes is better represented by nonrotating convection than geostrophic convection. As a result, we use scaling laws obtained in the idealized limit of nonrotating turbulent convection to make predictions about δ, pi < p*. In other words, we write δ = δt + δS with δt as the thickness of the turbulent boundary layer and δS as the thickness of the stable layer. We obtain δS as the positive solution of the equation Td(pi + ρ0gδS/104) = Tf(pi) + δSF/k, i.e., δS is found as the location z = − δS where the temperature of maximum density equals the temperature of the conductive base-state profile. The temperature at the base of the stable layer is correspondingly TS = Tf + δSF/k. The definition of the control parameter FRa , such that we assume heff = h − δS. Estimating αeff inside a convective lake is difficult since the vertical profiles of temperature and α are unknown. Here, for simplicity, we use αeff = α, i.e., we take α on the bottom boundary as the effective thermal expansion coefficient but assume that the temperature of the lake does not exceed TS. This assumption is likely to underestimate αeff but is the best conservative assumption possible without prior knowledge of the vertical temperature profile.First, we remark that the conductive layer at the top of the lake includes the turbulent boundary layer and a stable layer where α < 0 for eter RaF  of subglNu and the Reynolds number Re as functions of Ra inferred from recent state-of-the-art numerical simulations 7/9 and predict δt = 0.5heff/Nu = 0.5heff/(0.16Ra2/7), such thatCombining Assuming a conductive temperature profile in the stable and turbulent boundary layers, we then findRe = Uheff/ν and The turbulent flow velocity is inferred from The estimate of the characteristic turbulent length scale, or distance between plumes, is finally obtained from equation (6.3) of reference  and Ulsc = νRe/heff. The scaling laws for Nu and Re combine the expressions derived in subregions Iu, IIIu, and IVu of the GL theory [see  with the ice pressure  by decreasing the aspect ratio.Note that


These sites belong to two functional subclasses according to mechanistic criteria. We have also unveiled additional acetylation sites potentially involved in either the stimulation (Lys782) or the downmodulation  of specific Vav1-dependent downstream responses. Collectively, these results indicate that Nε-lysine acetylation can play variegated roles in the regulation of Vav1 signaling. Unlike the case of the tyrosine phosphorylation step, this new regulatory layer is not conserved in other Vav family paralogs.Vav1 works both as a catalytic Rho GTPase activator and an adaptor molecule. These functions, which are critical for T cell development and antigenic responses, are tyrosine phosphorylation-dependent. However, it is not known whether other posttranslational modifications can contribute to the regulation of the biological activity of this protein. Here, we show that Vav1 becomes acetylated on lysine residues in a stimulation- and SH2 domain-dependent manner. Using a collection of both acetylation- and deacetylation-mimicking mutants, we show that the acetylation of four lysine residues (Lys Nε-lysine acetylation is a posttranslational modification that was discovered on histones in 1964 ,3. HowevThe Vav family is a group of signal transduction proteins that work both as guanosine nucleotide exchange factors (GEFs) for Rho GTPases and adaptor molecules. This family has single representatives in invertebrates and three members in most vertebrate species  ,5. These2+-dependent stimulation of NFAT . A regions A.Δ835−845). The latter protein displays, similarly to Vav1Δ1−186, constitutive, tyrosine phosphorylation-independent biological activity due to the lack of the autoinhibitory CSH3 domain [Δ1−186 [Δ835−845 is capable of activating the NFAT pathway since it still keeps the N-terminal CH domain [WT but not of Vav1Δ835−845) or to subsequent effects in the effector signaling phases of the activated protein .The stimulation of the NFAT pathway by Vav1 is mediated by a GTPase-independent, adaptor-like mechanism that involves the phospholipase Cγ-mediated stimulation of the nuclear translocation and activation of this transcriptional factor A. The op3 domain . However [Δ1−186 ,24,32, VH domain . The use(i) that the Lys to Arg or the Lys to Gln mutant versions of Vav1 on those sites elicited changes in either the NFAT or JNK activity when compared to the WT counterpart; (ii) that those effects were opposite in the case of the Lys to Arg and Lys to Gln mutants ; and (iii) although prima facie counterintuitive, we also deemed as potentially relevant sites those in which the Lys to Arg and Lys to Gln mutations elicited the same effect on the biological activity of the mutant protein. The inclusion of these sites was based on the fact that those two types of mutations can elicit the same effect if they target lysyl groups that perform a key intramolecular role in the protein that cannot be replaced by the side group of the newly incorporated mutant residue. This scenario was contemplated given our previous experience with the characterization of the key regulatory tyrosine phosphorylation sites of Vav proteins. Indeed, during these analyses, we found that the dephosphorylation- (Tyr to Phe) and phosphorylation-mimicking (Tyr to Glu) mutations elicited in fact the same activation effect on the mutant proteins [We considered that a lysine acetylation site was functionally relevant when it fulfilled two criteria: proteins ,21,24,33335 and Lys374. These residues are located within the catalytic site of the DH domain . Similar data were observed when these mutants were tested in other catalysis-dependent readouts such as the stimulation of serum-responsive factor and the AP1 family (data not shown). This is probably connected with the reduced catalytic output of these mutant proteins, as assessed by the determination of Vav1-driven levels of GTP-bound Rac1 in cells using G-LISA® methods  present in a region of the DH domain that is located opposite to the catalytic site  it shows high levels of acetylation in cells according to our mass spectrometry determinations  iinations ; and  and Vav3 (Lys717 residue)  , we nextresidue) . Moreoveresidue) . Interesresidue) E, we coun Vav3WT . Thus, dn Vav3WT , the LysK587Q (rescue of activity) and Vav1K716Q (no rescue) mutants, we decided to investigate whether the enforced membrane localization of Vav1 could restore normal NFAT activity levels in the case of the mutant Vav1K222Q+K252Q protein  and Lys to Gln (K3xQ) mutations in those sites. We found that the ectopically expressed Vav1K3xR mutant displays higher levels of NFAT activity in both nonstimulated and stimulated cells when compared with the wild-type counterpart .Finally, we analyzed the potential contribution of the Lys of view , this is of view A,B. Give protein , it is l335, Lys716, and Lys782) that display much higher acetylation rates   B. It is protein) .Subclass A: The acetylation of the sites belonging to this subclass leads to the specific downmodulation of Vav1-mediated activation of NFAT while it preserves the stimulation of the Rac1-JNK axis. Unlike the rest of residues interrogated in this study, we have observed that the acetylation- and deacetylation-mimicking mutations of residues of subclass A elicit different effects on the biological activity of Vav1 , the KRR (Lys587), and the SH2 (Lys716) domains  and the DH domain , seems to be associated with the regulation of the stability of the protein at the plasma membrane. Consistent with this, the inclusion of the C-terminal membrane anchoring signal restores normal signaling levels in Vav1 proteins that bear acetylation-mimetic mutations in those sites. Therefore, the regulatory role of these acetylation sites is similar to the recently described Vav1 C1-KRR phosphatidylinositol 5-phosphate binding motif [587 is probably the most relevant of those sites as judged by the dire impact that its acetylation-mimetic mutation induces on the stimulation levels of the Vav1-NFAT pathway (716) is plasma membrane-independent. Our data also indicate that the role of this site is not related with the regulation of the optimal interaction of Vav1 with upstream tyrosine kinases, at least under cell stimulation conditions . The effect of both the deacetylation- and acetylation-mimicking mutations tested for those two sites is the reduction, although not full abolition, of the Rac1-JNK axis  and NFAT-dependent  responses in mature T cells. By contrast, they would preserve other functions directly associated with the catalysis-dependent pathways such as the stimulation of Rac1 itself, of the actin polymerization, of the Akt and integrin pathways, and the expression of NFAT-independent gene expression programs. The effect of the acetylation of the lysine residues that have been assigned to the subclasses B and C is more difficult to forecast given that, for example, variations in the strength of the catalytic output of the protein can elicit antagonistic effects during the positive and negative selection of immature T cells. In the case of mature T cells, we surmise that such acetylation steps would result in either the enhancement (subclass-B) or reduction (subclass-C) of the catalysis-dependent responses of Vav1 described above. Ultimately, the final outcome of these acetylation steps will be highly dependent on both the amount and functional significance of the pool of Vav1 molecules that undergoes this posttranslational modification during the stimulation cycle of both immature and mature T lymphocytes.Vav1 has been primarily involved in the positive and negative selection of thymocytes as well as in the effector functions of mature T cells ,17,52,53Our study indicates that the acetylation residues of Vav1 show low levels of phylogenetic  and func


Mycobacterium tuberculosis complex (MTBC) genomes suggests their most recent common ancestor followed human migrations out of Africa approximately 70,000 years before present. However, studies using ancient genomes as calibration points have yielded much younger dates of less than 6000 years. Here, we aim to address this discrepancy through the analysis of the highest-coverage and highest-quality ancient MTBC genome available to date, reconstructed from a calcified lung nodule of Bishop Peder Winstrup of Lund (b. 1605–d. 1679).Although tuberculosis accounts for the highest mortality from a bacterial infection on a global scale, questions persist regarding its origin. One hypothesis based on modern M. tuberculosis genome. In utilizing this high-quality, high-coverage seventeenth-century genome as a calibration point for dating the MTBC, we employed multiple Bayesian tree models, including birth-death models, which allowed us to model pathogen population dynamics and data sampling strategies more realistically than those based on the coalescent.A metagenomic approach for taxonomic classification of whole DNA content permitted the identification of abundant DNA belonging to the human host and the MTBC, with few non-TB bacterial taxa comprising the background. Genomic enrichment enabled the reconstruction of a 141-fold coverage The results of our metagenomic analysis demonstrate the unique preservation environment calcified nodules provide for DNA. Importantly, we estimate a most recent common ancestor date for the MTBC of between 2190 and 4501 before present and for Lineage 4 of between 929 and 2084 before present using multiple models, confirming a Neolithic emergence for the MTBC. Mycobacterium tuberculosis complex (MTBC), has taken on renewed relevance and urgency in the twenty-first century due to its global distribution, its high morbidity, and the rise of antibiotic-resistant strains [Tuberculosis, caused by organisms in the  strains . The dif strains , makes tMycobacterium tuberculosis data. One estimate suggests the extant MTBC emerged through a bottleneck approximately 70,000 years ago, coincident with major migrations of humans out of Africa [M. tuberculosis genomes, with internal nodes of the MTBC calibrated by extrapolated dates for major human migrations [M. tuberculosis genomes from the late eighteenth and early nineteenth centuries [Existing literature suggests two estimates for the time to the most recent common ancestor (tMRCA) for the MTBC based on the application of Bayesian molecular dating to genome-wide f Africa . This esgrations . This esgrations . As an agrations . This yoenturies .Mycobacterium pinnipedii, a lineage of the MTBC currently associated with infections in seals and sea lions [Despite the agreement in studies that have relied on ancient DNA calibration so far, dating of the MTBC emergence remains controversial. The young age suggested by these works cannot account for purported detection of MTBC DNA in archeological material that predates the tMRCA estimate  scans of the mummified remains of Bishop Peder Winstrup of Lund, Sweden revealed a calcified granuloma a few millimeters (mm) in size in the collapsed right lung together with two ~ 5 mm calcifications in the right hilum Fig. . PrimaryHomo sapiens. A further 1724 reads were assigned to the Mycobacterium tuberculosis complex (MTBC) node. Importantly, no other taxa in the genus Mycobacterium were identified, and the only other identified bacterial taxon was Ralstonia solanacearum   using BWDue to the clear but low-abundance MTBC signal, a uracil DNA glycosylase (UDG) library was constructed to remove DNA lesions caused by hydrolytic deamination of cytosine residues  and enriWe further evaluated the quality of the reconstructed genome by quantifying the amount of heterozygous positions see the “” sectionPreliminary phylogenetic analysis using neighbor joining  tree was generated for the BDSKY+UCLD model, with 3258 years before present (BP)  as an estimated date of emergence for the MTBC Fig. . Date raR2 = 0.113; see the “The L4 dataset includes LUND1 and the two Hungarian mummies described above  as calibWe applied the same models as described above for the full MTBC dataset, with the addition of a birth-death skyline model conditioned on the origin of the root (BDSKY+UCLD+origin). All mean tree heights are within 250 years of each other and the 95% HPD intervals largely overlap. The BDSKY+UCLD and BDSKY+UCLD+origin models show the highest marginal likelihood values after stepping stone sampling. We employed the BDSKY+UCLD+origin model to determine if the estimated origin of the L4 dataset agreed with the tree height estimates for the full MTBC dataset. Intriguingly, the estimated origin parameter Table , or the R2 value for the relationship between sampling time and root-to-tip distance as calculated using TempEst, we also performed a date randomization test of the L4 BDSKY+UCLD model, in which we shuffled the sampling dates randomly among all genomes [A calibrated MCC tree Fig.  was gene genomes , 41. We Mycobacterium tuberculosis genomes is steadily reducing the uncertainty of molecular dating estimates for the emergence of the MTBC. Here, using the ancient data available to date, we directly calibrate the MTBC time tree and confirm that known diversity within the complex is derived from a common ancestor that existed ~ 2000–6000 years before present  that may have been subject to dramatically different evolutionary pressures compared to the human-associated lineages of the complex due to differing host biology and population dynamics. However, our use of a relaxed clock model allowed for the estimation and accommodation of variable rates across different branches of the complex. We do not see evidence for divergent substitution rates among the branches leading to the Peruvian M. pinnipedii strains . We chose rho priors  derive from an Old World context  pre-dating the White Plague in Europe or (b) come from any geographical location or pre-modern time period, but belong to one of the MTBC lineages not yet represented by ancient data. An ideal data point, which would clarify many open questions and seeming contradictions related to the evolutionary history of the MTBC, would derive from Africa, the inferred home of the MTBC ancestor , 43–45, M. tuberculosis genomes, and the refinement and improved parameterization of Bayesian models that reflect the realities of MTBC (and other organisms’) population dynamics and sampling frequencies over time. To aid in future attempts to answer these questions, this study provides an ancient MTBC genome of impeccable quality and explores the first steps in applying birth-death population models to modern and ancient TB data.Here, we offer confirmation that the extant MTBC, and all available ancient MTBC genomes, stem from a common ancestor that existed a maximum of 6000 years before present. Many open questions remain, however, regarding the evolutionary history of the MTBC and its constituent lineages, as well as the role of tuberculosis in human history. Elucidating these questions is an iterative process, and progress will include the generation of diverse ancient The paleopathological investigation of the body of Winstrup is based on extensive CT scan examinations with imaging of the mummy and its bedding performed with a Siemens Somatom Definition Flash, 128 slice at the Imaging Department of Lund University Hospital. Ocular inspection of the body other than of the head and hands was not feasible, since Winstrup was buried in his episcopal robes and underneath the body was wrapped in linen strips. The velvet cap and the leather gloves were removed during the investigation. The body was naturally mummified and appeared to be well preserved with several internal organs identified.The imaging was quite revealing. The intracranial content was lost with remains of the brain in the posterior skull base. Further, the dental status was poor with several teeth in the upper jaw affected by severe attrition, caries, and signs of tooth decay, as well as the absence of all teeth in the lower jaw. Most of the shed teeth were represented by closed alveoli, indicating antemortem tooth loss. Along with the investigation of the bedding, a small sack made of fabric was found behind the right elbow containing five teeth: two incisors, two premolars, and one molar. The teeth in the bag complemented the remaining teeth in the upper jaw. It is feasible that the teeth belonged to Winstrup and were shed several years before he died. A fetus approximately 5 months of age was also found in the bedding, underneath his feet.Both lungs were preserved but collapsed with findings of a small parenchymal calcification and two ~ 5 mm calcifications in the right hilum Fig. . The assThe skeleton showed several pathological changes. Findings on the vertebrae consistent with DISH  were present in the thoracic and the lumbar spine. Reduction of the joint space in both hip joints and the left knee joint indicate that Winstrup was affected by osteoarthritis. No signs of gout or osteological tuberculosis  were found.Neither written sources nor the modern examination of the body of Winstrup reveal the immediate cause of death. However, it is known that he was bedridden for at least 2 years preceding his death. Historical records indicate that gallstones caused him problems while traveling to his different parishes. Additionally, he was known to have suffered from tuberculosis as a child, which may have recurred in his old age.Sampling of the lung nodule, extraction, and library preparation were conducted in dedicated ancient DNA clean rooms at the Max Planck Institute for the Science of Human History in Jena, Germany. The nodule was broken using a hammer, and a 5.5 mg portion of the nodule was taken with lung tissue for extraction according to a previously described protocol with modifications . The sam2O, 5 μl of NEB Buffer 2 (New England Biolabs), 2 μl dNTP mix (2.5 mM), 4 μl BSA (10 mg/ml), 5 μl ATP (10 mM), 2 μl T4 polynucleotide kinase, and 0.4 μl T4 polymerase, then purified and eluted in 18 μl TET. Illumina adapters were ligated to the blunt-end fragments in a reaction with 20 μl Quick Ligase Buffer, 1 μl of adapter mix (0.25 μM), and 1 μl of Quick Ligase. Purification of the blunt-end repair and adapter ligation steps was performed using MinElute columns (Qiagen). Adapter fill-in was performed in a 40-μl reaction including 20 μl adapter ligation eluate, 12 μl H2O, 4 μl Thermopol buffer, 2 μl dNTP mix (2.5 mM), and 2 μl Bst polymerase. After the reaction was incubated at 37 °C for 20 min, the enzyme was heat deactivated with a 20-min incubation at 80 °C. Four library blanks were processed alongside LUND1 to control for reagent/laboratory contamination. The library was quantified using a real-time qPCR assay (Lightcycler 480 Roche) with the universal Illumina adapter sequences IS7 and IS8 as targets. Following this step, the library was double indexed [2O, 10 μl PfuTurbo buffer, 1 μl PfuTurbo (Agilent), 1 μl dNTP mix (25 mM), 1.5 μl BSA (10 mg/ml), and 2 μl of each indexing primer (10 μM). The master mix was prepared in a pre-PCR clean room and transported to a separate lab for amplification. The two reactions were purified and eluted in 25 μl of TET each over MinElute columns (Qiagen), then assessed for efficiency using a real-time qPCR assay targeting the IS5 and IS6 sequences in the indexing primers. The reactions were then pooled into one double-indexed library. Approximately one third of the library was amplified over three 70 μl PCR reactions using 5 μl of template each and Herculase II Fusion DNA Polymerase (Agilent). The products were MinElute purified, pooled, and quantified using an Agilent Tape Station D1000 Screen Tape kit. LUND1 and the corresponding negative controls were sequenced separately on an Illumina NextSeq 500 using single-end, 75-cycle, high-output kits.Double-stranded Illumina libraries were constructed according to an established protocol with some modifications . Overhan indexed  with a uDe-multiplexed sequencing reads belonging to LUND1 were processed in silico with the EAGER pipeline (v.1.92) . ClipAndSingle-stranded probes for in-solution capture were designed using a computationally extrapolated ancestral genome of the MTBC . The pro2O, 10 μl NEB Buffer 2 (New England Biolabs), 12 μl dNTP mix (2.5 mM), 1 μl BSA (10 mg/ml), 10 μl ATP (10 mM), 4 μl T4 polynucleotide kinase, and 6 μl USER enzyme (New England Biolabs). The reaction was incubated at 37 °C for 3 h, and then 4 μl of T4 polymerase was added to the library to complete the blunt-end repair step. The remainder of the library preparation protocol, including double indexing, was performed as described above.Fifty microliters of the original LUND1 extract were used to create a uracil-DNA glycosylase (UDG) treated library, in which the post-mortem cytosine to uracil modifications, which cause characteristic damage patterns in ancient DNA, are removed. The template DNA was treated in a buffer including 7 μl HM. tuberculosis DNA also underwent capture in parallel with the LUND1 libraries. Capture was performed according to an established protocol [The LUND1 UDG-treated library was amplified over two rounds of amplification using Herculase II Fusion DNA Polymerase (Agilent). In the first round, five reactions using 3 μl of template each were MinElute purified and pooled together. The second round of amplification consisted of three reactions using 3 μl of template each from the first amplification pool. The resulting products were MinElute purified and pooled together. The final concentration of 279 ng/μl was measured using an Agilent Tape Station D1000 Screen Tape kit (Agilent). A portion of the non-UDG library (see above) was re-amplified to 215 ng/μl. A 1:10 pool of the non-UDG and UDG amplification products was made to undergo capture. A pool of all associated negative control libraries  [M. tuberculosis infection. Sites with missing or incomplete data were excluded from further analysis.For the enriched, UDG-treated LUND1 sequencing data, de-multiplexed paired-end reads were processed with the EAGER pipeline (v. 1.92) , adapternalyzer)  to creatnalyzer) , with renalyzer) . Potentinalyzer)  separatenalyzer)  as inputNeighbor joining , as all ancient calibration points were limited to Lineage 4 .Additional file 6: Table S5. List of genomes included in the L4 dataset, with respective publications, accession numbers, lineages, dates, and percentage of total SNPs called as heterozygous.Additional file 7: Table S6. List of sites excluded from the full MTBC dataset in GFF format.Additional file 8: Table S7. List of sites excluded from the L4 dataset in GFF format.Additional file 9: Table S8. SnpEff annotation for derived alleles in LUND1.Additional file 10: Table S9. Date randomization results for MTBC dataset.Additional file 11: Table S10. Date randomization results for L4 dataset.Additional file 12. Review history.


Precise pedicle screw placement is a critical skill during minimally invasive spinal surgeries but can pose various challenges. Techniques such as electromyography (EMG) have been traditionally utilized for this purpose but have several shortcomings. Transabdominal motor action potential (TaMAP) has been examined as a possible effective neuromonitoring alternative and is hypothesized to provide important data on symptomatic malpositioned pedicle screws. The current study seeks to determine whether TaMAP may be an advantageous technique in the neuromonitoring of percutaneous pedicle screw placement during minimally invasive spinal procedures. The methodology involved recording TaMAP signals at the outset and the conclusion of spinal surgical procedures in human participants, for which comparisons were made of pre- and post-operative data. Results revealed that TaMAP signals remained stable during accurate pedicle screw placement and degraded during a case of inaccurate placement, for which initial misplaced hardware altered the depolarization threshold and resulted in substantial signal alteration. These results suggest that TaMAP, which is stable, repeatable, and reflects real-time information, can potentially be used as a reliable and more precise indication of accuracy in pedicle screw placement during spinal surgeries. This is the first TaMAP study conducted in human participants. BackgroundPrecise placement of percutaneous pedicle screws is a critical technique for minimally invasive spinal procedures . AlthougIntraoperative spinal procedures utilizing pedicle screws can treat a variety of conditions causing back pain such as spinal trauma, degenerative spinal diseases, scoliosis, and spondylolisthesis . In miniInjuries during pedicle screw placementAttempts to accurately position pedicle screws can pose several challenges. For example, the corridor of the pedicle is narrow and can damage adjacent vital structures when breached . StudiesDrawbacks to the use of EMG alone during screw placementPrior studies demonstrated the effectiveness of using EMG responses generated by electrical stimulation. One such study, which was conducted in the abdominal and leg muscles of sheep, showed that EMG could be effectively used to indicate the pedicle screw position . EMG hasTaMAP as a neuromonitoring techniqueTaMAP is a relatively new form of neurophysiological monitoring that has shown effectiveness in detecting inadvertent iatrogenic nerve damage due to spinal procedures . This inNeuromonitoring was conducted during the placement of percutaneous pedicle screws. Out of the first 50 patients who underwent minimally invasive spinal procedures, only six underwent fusion procedures involving pedicle screw placement, including extreme lateral interbody fusion (XLIF), transforaminal lumbar interbody fusion (TLIF), and posterior spinal fusion (PSF). These patients were subjected to TaMAP recording in conjunction with routine EMG monitoring. All patients who underwent percutaneous pedicle screw placement prior to a minimum three-month follow-up were included in the analysis. Baseline TaMAPs were acquired prior to incision, and final responses were again examined upon wound closure. Data were obtained from preoperative and postoperative neurological exams . Responses were measured by the level of current required to depolarize the nerve and subsequent muscle . All patients showed detectable TaMAPs that were stable and repeatable during the procedure. Stimulation currents over 600 mAmps were observed in patients with increased body mass index (BMI) and in deeper muscle recordings. Action potential for individual muscle groups was monitored for amplitude and latency. The TaMAP depolarization value remained unchanged, with appropriate pedicle screw placement in any of the patients. In a single case, an L4 pedicle screw was misplaced medially, and an increase in depolarization in that root was identified. Notably, after the screw was replaced in the appropriate location, the stimulation value returned to normal.According to results, out of the six patients who received minimally invasive spinal fusion procedures involving pedicle screw placement for the aforementioned muscle groups, only one pedicle screw was inaccurately placed, resulting in a drastic activity spike. As seen in Figure 2, the pedicle screw was misplaced for the right tibialis anterior, resulting in a recorded measurement of 1128 mA. When the screw was replaced in the correct position, the signal returned to the normal value. This is in concordance with our predictions, as misplaced pedicle screws would be expected to result in an increased value if the nerve is impinged, given that an irritated nerve requires more energy to reach depolarization . Thus, oThe use of this monitoring system is relatively safe and requires no change in anesthetic technique, as it reflects a peripheral response , other than avoiding paralytic agents. Although EMG methods, such as triggered EMG (trEMG), involve stimulating the pedicle directly with an electric probe -21, the Additional studies are needed to further corroborate these findings and continue to examine the benefits of this technique. There is currently only one other study to our knowledge discussing TaMAP, as it is a relatively novel tool . Given tNeuromonitoring during the placement of pedicle screws, especially those placed percutaneously, has recently become more standard. The current study suggests a unique application for the novel transabdominal motor action potential procedures during minimally invasive spinal procedures. This technique allows the surgeon to identify nerve compression, which may be symptomatic but missed by other modalities such as EMG alone. The current study provides support for the possibility of TaMAP as a useful neuromonitoring technique in minimally invasive spinal surgeries and is the first TaMAP study conducted in human participants.


In the early 1930s, infectious bronchitis (IB) was first characterized as a respiratory disease in young chickens; later, the disease was also described in older chickens. The etiology of IB was confirmed later as being due to a coronavirus: the infectious bronchitis virus (IBV). Being a coronavirus, IBV is subject to constant genome change due to mutation and recombination, with the consequence of changing clinical and pathological manifestations. The potential use of live attenuated vaccines for the control of IBV infection was demonstrated in the early 1950s, but vaccine breaks occurred due to the emergence of new IBV serotypes. Over the years, various IBV genotypes associated with reproductive, renal, gastrointestinal, muscular and immunosuppressive manifestations have emerged. IBV causes considerable economic impacts on global poultry production due to its pathogenesis involving multiple body systems and immune suppression; hence, there is a need to better understand the pathogenesis of infection and the immune response in order to help developing better management strategies. The evolution of new strains of IBV during the last nine decades against vaccine-induced immune response and changing clinical and pathological manifestations emphasize the necessity of the rational development of intervention strategies based on a thorough understanding of IBV interaction with the host. Coronaviridae and order Nidovirales [Infectious bronchitis virus (IBV) belongs to the family ovirales . IBV infovirales . The infovirales ,4,5,6. Yovirales . In addiovirales . Howeverovirales .The first IB case caused by the Mass serotype was recorded from North Dakota, USA . Since tThe variability of the spike (S)1 amino acid sequence determines the host and tissue tropism of IBV ,27 sinceThe evolution and occurrence of novel strains of IBV is a continuing process . AlthougIBV is typically transmitted to the host by inhalation, whereupon it attaches to the respiratory epithelium and enters by receptor-mediated endocytosis . Once IBIt is well established that, following the initial infection in the respiratory tract, the virus is disseminated to other tissues due to viremia ,57 Figu. HoweverSubsequent clinical manifestations of IBV infection related to the reproductive tract depend on the infecting IBV strain. For example, the M41, Aust T and QX-like strains are known to cause reproductive tract defects in long-lived chickens ,17,25, lHowever, IBV replication in reproductive mucosa has been documented in several studies ,61,62. DHistologically, deciliation of the epithelium and a reduction in the height of the epithelial cells, along with epithelial desquamation and degeneration, are common in infected oviducts between 7 and 21 dpi  with occAlthough the consequences of IBV targeting the reproductive tracts of female chickens is known, little attention has been given to changes corresponding to the reproductive tracts of male chickens, which may cause impacted sperm production, infertility and venereal transmission of IBV ,70. ReceThe nephropathogenic IBV strains include B1648, Aus T, QX-like, 4/91, Holte and Gray ,25,32,50IBV strains that target the kidney have received increased attention due to their higher virulence in young chickens when compared to other IBV strains ,32,73. AAt post-mortem examination, IBV-infected kidneys are pale, discolored and enlarged ,47. UratAlthough IBV has been isolated from cloacal swabs, there is no indication that IBV is transmitted via the fecal–oral route. It is possible that gastrointestinal infection follows respiratory infection, subsequent infection of monocytes and macrophages and the spread of IBV via the blood or lymphatics ,59. IBV In the early 1990s, broiler chickens infected with IBV strain 793/B were found to develop bilateral pectoral myopathy . The disDepending on the IBV strain, the virus can persist in the tissues of chickens for an extended period ,74,78. FThe IBV-induced potential immune-suppressive mechanisms are summarized in One of the immune cell types that bridges innate and adaptive host responses is the macrophages, and the available data show that certain IBV serotypes  target respiratory tract macrophages and replicate within them, thus leading to a productive infection ,88. AlthThe complement system functions as a part of the innate arm of the immune response and also plays a role in adaptive immune functions. The components of the complement pathway aid in the host immune response via complement- and antibody-mediated lysis of viruses ,93,94,95Mycoplasma gallisepticum followed by IBV infection can result in coryza, tracheitis and airsacculitis in the host [Escherichia coli can lead to prominent lesions in respiratory surfaces, pericarditis and death [Haemophilus paragallinarum with IBV could result in severe lesion development and increased mortality rates [IBV replication impacting the respiratory tract brings forth a defective clearance mechanism by the deciliation of the respiratory epithelium, thereby increasing the vulnerability of respiratory surfaces to secondary bacterial infections . The co-the host . In addind death . In addity rates .Protection against IB is mediated by both antibody- and cell-mediated immune responses ,103. AltVaccination is the primary choice for the control of IB in the poultry industry. The chickens receive multiple IB vaccinations, and the frequency of vaccination depends on the expected production life of the chicken. For broilers, day-old vaccination in the hatchery is commonly practiced followed by a booster vaccination at 2–3 weeks of age . MultiplHowever, vaccination of day-old chickens is controversial for three reasons. First, maternal antibodies could be protective against potential IBV infection during the first few days of life while early vaccination could enhance the decay of the maternal antibodies . Second,Since the introduction of vaccination against IB using live attenuated (Mass serotype) vaccines, different vaccine serotypes such as 4/91, Ark, Conn, D274 and D1466 have been developed and made available commercially . Based oAlthough control of IB relies on vaccination , severalGiven the limited efficacy of existing IB vaccination strategies, it is critical to establish an IBV surveillance system that characterizes the different IBV strains circulating in various geographical areas and that we understand the antigenic and/or genetic similarities between the circulating IBVs and the available IB vaccines. These data will lead to the optimization of IB vaccination strategies to prevent vaccine breaks. If the existing vaccines are not useful in optimizing vaccination strategies, it would also worth developing autogenous vaccines (inactivated) using characterized unique IBV isolates prevalent in a given geographical area in order to include in the existing vaccination regimes. Given the available scientific evidence against IB vaccination at the hatchery ,110, it After decades of research into IBV and numerous studies on vaccine efficacy for the control of IB, this disease is still a major economic concern globally. Although many studies have been conducted on IBV pathogenesis, there is little specific information on IBV receptors that determine macrophage tropism and tissue tropism, mechanisms that allow IBV dissemination from the respiratory tract to secondary tissues, IBV persistence in the cecal tonsils and kidney and the immunopathogenesis and immunosuppressive mechanisms of different strains of IBV. Novel and sensitive assays and other necessary tools are now available for in-depth investigations of the mechanisms involved in these pathogenesis events. Given the concern of vaccine breaks and the emergence of heterogeneous IBV strains, investigations leading to an in-depth understanding of the pathogenesis of IBV are necessary.Studies of host–IBV interaction lead to the understanding of tropism of IBV for various body systems, severity of lesions produced in each of these tissues and ways the virus is shed to the environment ,64,134. In addition to secondary bacterial infections, vaccine induced immune response to any pathogen can be impacted by immune suppression induced by IBV. It is necessary to determine if IBV induced immune suppression impact the immune responses intended to be generated by a variety of vaccines used in chickens . Another


Lactuca sativa “Great Lakes 366”) seedling growth using the sandwich and dish pack methods, respectively. With the sandwich method, parsley (Petroselinum sativum) showed the strongest inhibitory effect on lettuce radicle growth (77%), followed by tarragon (Artemisia dracunculus) (72%). However, caraway (Carum carvi), dill (Anethum graveolens) (seed), laurel (Laurus nobilis), rosemary , and sage  were the most inhibitory species  in the dish pack method. Cardamom (Elettaria cardamomum) and thyme (Thymus vulgaris) also showed strong inhibitory activity . The headspace sampling and gas chromatography-mass spectrometry (GC-MS) analysis identified the main inhibitory active compounds as carvone in caraway and dill (seeds), 1,8-cineole in laurel and cardamom, and borneol in thyme. Both camphor and 1,8-cineole were detected in rosemary and sage, and the total activity evaluation showed that camphor was the major inhibitory compound in rosemary, although both compounds played equal roles in sage.The inhibitory activities of the leachates and volatiles from 53 plant species (spices and herbs) were evaluated against lettuce ( A range of secondary metabolites is synthesized by plants, with the exact composition varying among species. Application of these compounds as agricultural chemicals has been well investigated, with many of the insecticides and fungicides that are used in recent years originating from natural plants . The inhElwendia persica). Allelochemicals are released from plants into the environment through several routes, including volatilization from the leaf tissues, leaching of non-volatiles from the foliage by rainfall, exudation from living roots, or the decomposition of residues by soil microorganisms ,12,13. STherefore, in this study, we evaluated the growth inhibitory activities of the leachates and volatiles of 53 plant species (spices and herbs) using the sandwich and dish pack methods, respectively, and determined the active component(s) responsible for any such inhibitory effects.In this study, we examined the plant growth inhibitory activities of 53 species of plants (spices and herbs) against the growth of lettuce seedlings using the sandwich and dish pack methods. The volatile compounds responsible for the activities of the most inhibitory species were also identified. Lettuce was chosen as the test plant because it germinates quickly with high uniformity and has a high sensitivity to allelochemicals . In addiOxalis corniculata, Rumex acetosella, and Begonia spp., showed 90% to 95% inhibition when lettuce was treated with 10 mg of plant samples in the sandwich method. Using the same amount of sample, we found that none of the species tested exhibited > 90% growth inhibition with the sandwich method (Petroselinum sativum) showing the strongest inhibition (77%), followed by tarragon (Artemisia dracunculus) (72%). It has previously been reported that parsley contains myristicin and apiole both of which have shown inhibitory activities against the seedling growth of rice [Artemisia, including tarragon, have been reported to be allelopathic [Eugenia aromatica) showing the strongest inhibition (72%), followed by oriental mustard (Brassica juncea) (69%). However, seven of the evaluated samples showed a stimulatory growth effect, with seri roots (Oenanthe javanica) and red shiso (Perilla frutescens), in particular, promoting growth by >20%. Sekine et al. [Fusarium oxysporum using the dish pack method. Using the same amount of sample, we found that caraway (Carum carvi), dill seeds (Anethum graveolens), laurel (Laurus nobilis), rosemary , and sage  caused complete inhibition of lettuce seed growth in all five wells  and thyme (Thymus vulgaris), also caused 100% inhibition in three of the five wells. Additionally, the pepper tree (Schinus molle) and mace (Myristica fragrans) caused strong inhibition in the two 41 mm distance wells. However, their activity sharply decreased with increasing distance  from the source well, indicating low volatility of the allelopathic compounds produced by these species. By contrast, the green and red varieties of shiso (Perilla frutescens) stimulated the radicle growth of lettuce by two-fold as compared with the control. It has been reported that shiso leaves contain the aromatic compound pellylaldehyde [Other species including cardamom , laurel, rosemary, sage, cardamom, and thyme) were determined through headspace sampling and GC-MS analysis. The GC-MS analysis resulted in the detection of 12 compounds . Among tZea mays) in the order of camphor > 1,8-cineole > α-pinene > limonene. Evaluation of allelopathic activities of the authentic samples of the 12 detected compounds showed that borneol, camphor, carvone, and 1,8-cineole resulted in 100% growth inhibition in all of the wells. In addition, 3-carene and β-pinene exhibited high levels of inhibition when 50 μL of the compounds were used . FurtherBrassica campestris was inhibited by these compounds and β-pinene in the order of camphor > 1,8-cineole > β-pinene > α-pinene > camphene. The inhibition trends reported in both studies are similar to our findings despite the use of different types of receptor plants. Contrary to the results of this study where borneol showed higher activity than carvone, Vokou et al. [50) and total activity. Evaluation of the specific activity  expressed as the EC50 and essential for the development of pesticides, as a compound that exhibits a small EC50 value has a high specific activity. By contrast, the evaluation by total activity  is important for biological use [Spiraea thunbergii through the concept of total activity. The EC50 values of authentic camphor and 1,8-cineole were 0.0633 ppm and 7.21 ppm, respectively. The total activity, based on these values and the concentration of the compounds, was calculated to be almost 10 times higher for camphor than for 1,8-cineole  in rosemary. Almost the same total activity for both compounds in sage  indicates that they play equal roles in the inhibitory activity of this herb.In another study, Nishida et al.  reportedu et al.  reportedical use ,44. Hiraical use  isolatedDried samples of 53 species of spices and herbs were tested for their potential allelopathy through leachates and volatiles . Thirty-Lactuca sativa “Great Lakes 366”, Takii Seed, Japan) seeds were placed on top of the gelatinized agar in the well. Three wells of a multi-dish were used as three replications of a single species. In addition, a control multi-dish was set up in the same manner only without the addition of any samples to the wells. The multi-dishes were incubated in a dark growth chamber at 25 °C for three days. The growth rate of the lettuce seedlings relative to the control was then measured to calculate the inhibition rate (control = 100% growth). The activity of the leachates produced by each plant sample was evaluated following the principles of the sandwich method using six-well multi-dishes  . Each weThe activity of the volatiles released by each plant sample was evaluated following the dish pack method procedure ,42 usingSeven lettuce seeds were placed on the surface of each of the remaining wells. The multi-dish was then covered, and the sides were sealed with adhesive tape to prevent volatile losses due to volatilization. In addition, a control dish was set up in the same manner only without the addition of any sample to the source well. The multi-dishes were incubated in a dark growth chamber at 25 °C for three days. The radicle and hypocotyl lengths of the lettuce seedlings in each well were measured. The inhibition rate was, then, calculated in the same manner as for the sandwich method see . There wThe volatiles that were released by the species showing the highest activities were determined using the headspace method. A finely ground sample (200 mg) of each species was put into a glass vial equipped with aluminium crimp seal cap with a polytetrafluoroethylene (PTFE)/silicone septum and was kept under laboratory conditions for 30 min. A headspace vapour sample (1 mL) was, then, taken from the vial through the septum using a Hamilton gas-tight syringe. The collected gas was then immediately injected into a QP-5050A gas spectrometer  using EQUITY-5 gas chromatography (GC) column . Each compound was identified by comparing its mass spectrum with values recorded in the NIST Mass Spectral Library and the retention time of an authentic sample on the GC. The quantity of each compound from a given plant sample was determined by comparing the peak area of the compound with that of its authentic sample. The operating conditions of the gas chromatographer-mass spectrometer were as follows: Inlet 200 °C and column oven 40 °C for 30 s and, then, programmed to increase by 8 °C/min to 160 °C, which was maintained for 5 min. The split-less injection was applied using 1.0 mL of vapour-phase sample or 2.0 μL of the liquid sample. Headspace sampling and analysis were not replicated.To evaluate the growth inhibitory activities of authentic samples of compounds that corresponded to the detected compounds, the same procedure was applied for testing the dried samples. The only change was a cup containing 50 μL of the authentic compound (or 50 mg for solid compounds) placed in the source well instead of the dried sample . Authent50), is the effective concentration of the compound that induces half-maximum inhibition. In our study, the EC50 value was calculated using a probit analysis [To measure the vapour concentration of the authentic compound in each well, separate multi-dishes were set out in the same manner as above but without lettuce seeds. One multi-dish corresponded to a well, and the vapour was collected from the multi-dish only once to avoid any unusual diffusion of vapour during the collection process. The vapour was collected after 24 h using a Hamilton gas-tight syringe (1.0 mL) through a septum located on top of the wells. The gas chromatography-mass spectrometry (GC-MS) analysis was undertaken using the same procedure as in analysis , and theThe leachates from parsley, followed by tarragon showed the strongest inhibitory activity against lettuce seedling growth using the sandwich method. On the other hand, the volatile constituents from caraway, dill seed, laurel, rosemary, and sage followed by cardamom and thyme showed the highest inhibition using the dish pack method. Headspace sampling and GC-MS analysis identified the main inhibitory compounds as carvone in caraway and dill seed, 1,8-cineole in laurel and cardamom, and borneol in thyme. Both camphor and 1,8-cineole were detected in rosemary and sage. Although both compounds played potentially equal roles in the inhibitory activity of sage, the total activity evaluation demonstrated that camphor was the major inhibitory compound in rosemary.


Angelica decursiva has been used for the treatment of many diseases. The goal of this study was to evaluate the potential of four natural major dihydroxanthyletin-type coumarins—(+)-trans-decursidinol, Pd-C-I, Pd-C-II, and Pd-C-III—to inhibit the enzymes, protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase. In the kinetic study of the PTP1B enzyme’s inhibition, we found that (+)-trans-decursidinol, Pd-C-I, and Pd-C-II led to competitive inhibition, while Pd-C-III displayed mixed-type inhibition. Moreover, (+)-trans-decursidinol exhibited competitive-type, and Pd-C-I and Pd-C-II mixed-type, while Pd-C-III showed non-competitive type inhibition of α-glucosidase. Docking simulations of these coumarins showed negative binding energies and a similar proximity to residues in the PTP1B and α-glucosidase binding pocket, which means they are closely connected and strongly binding with the active enzyme site. In addition, dihydroxanthyletin-type coumarins are up to 40 µM non-toxic in HepG2 cells and have substantially increased glucose uptake and decreased expression of PTP1B in insulin-resistant HepG2 cells. Further, coumarins inhibited ONOO−-mediated albumin nitration and scavenged peroxynitrite (ONOO−), and reactive oxygen species (ROS). Our overall findings showed that dihydroxanthyletin-type coumarins derived from A. decursiva is used as a dual inhibitor for enzymes, such as PTP1B and α-glucosidase, as well as for insulin susceptibility.As a traditional medicine,  Diabetes mellitus (DM) has risen quickly in recent decades and currently, over 415 million DM sufferers worldwide, predicted to hit 642 million by 2040 . Type 2 Protein tyrosine phosphatase 1B (PTP1B) is part of the intracellular PTP that is active in the negative regulation of insulin and leptin signaling systems . PTP1B cReducing postprandial hyperglycemia and slowing glucose absorption by inhibiting carbohydrate-hydrolyzing enzymes, such as α-glucosidase, is one of the therapeutic approaches to restore insulin sensitivity . α-Gluco‒ an action that can be indirectly inferred by nitrotyrosine residues in the production of ONOO‒ -2-deoxy-D-glucose (2-NBDG) was purchased from Life Technologies . Dithiothreitol (DTT) was purchased from Bio-Rad Laboratories . Fetal bovine serum (FBS), penicillin-streptomycin, minimum essential medium (MEM), 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA), nonessential amino acids, and sodium pyruvate were purchased from Gibco-BRL Life Technologies . β-Actin, secondary antibodies, and PTP1B were obtained from Santa Cruz Biotechnology . All other solvents and chemicals were purchased from E. Merck, Fluka, or Sigma-Aldrich, unless otherwise stated.A. decursiva was purchased from the Korean plant extract bank, which is affiliated with the Korea bioscience and biotechnology research institute, Korea. The whole plant has been documented and deposited for reference in the Department of Food Science and Nutrition, Pukyong National University, Busan, South Korea .Entire plants of A. decursiva (2.9 kg) was refluxed for 3 h (3 × 10 L) with methanol (MeOH). In order to produce the MeOH extract (763.32 g), the total filtrate was then concentrated until dried in vacuo at 40 °C. This extract was suspended in distilled water and subsequently divided into dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and n-butanol (n-BuOH) for the processing of CH2Cl2 (210.47 g), EtOAc (15.06 g) and n-BuOH (159.07 g) fractions, respectively, in addition to the water residue (444.32 g). Initially, the CH2Cl2 fraction (210.47 g) was subjected to silica gel column chromatography and eluted with CH2Cl2, followed by the addition of gradually increasing amounts of MeOH, to create 20 subfractions (CF01–CF20). Fraction 4 (CF04) has also been chromatographed over silica gel using hexane: EtOAc (10:1) in order to obtain three subfractions (CF05F1–CF05F3). Fraction 1 of fraction 5 (CF05F01) was chromatographed using CH2Cl2 over silica gel to obtain Pd-C-I (200 mg) and Pd-C-III (92 mg). Fraction 2 of fraction 5 (CF05F03) was also chromatographed over silica gel using CH2Cl2 to obtain Pd-C-II (42 mg). Fraction 6  was chromatographed with n-hexane:EtOAc  on a silica gel column to obtain (+)-trans-decursidinol (130.13 mg). All compounds were previously isolated and identified in our laboratory -1-naphthyl}-l-alanyl)amino]pentyl}oxy)-2-naphthoic acid (compound 23)  on ice after stimulation with 100 nM insulin at 37 °C for 30 min. Insoluble materials were extracted by centrifugation for 20 min at 25,000 × g after incubation for 30 min. An updated Bradford protein assay kit calculated the protein concentrations. Total protein (40 μg) was electrophoresed into a polyvinylidene difluoride (PVDF) membrane by use of a dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In a blocking buffer, membranes were blocked  and incubated overnight at 4 °C with primary antibodies, followed by incubation at room temperature for 4 h with suitable secondary antibodies. Three times for 30 min with TBST, the membranes were washed. The Supersignal West Pico chemiluminescence substrate  has been used to visualize the protein bands and pictured on X-ray film  according to the manufacturer’s instructions. Using CS analyzer software , bands were scanned and quantitated and the control was set to 1. As the internal control, β-actin was used. For determining the molecular weights of protein bands, prestained blue protein markers were used. Comparisons were made between mean band densities in same gels. Autoradiography visualized protein bands and measured the intensities using Quantity One software .Insulin-resistant HepG2 cells  and Duncan’s test  test were used to evaluate statistically significant values. A statistically significant pNPP and pNPG as a substrate to evaluate the anti-diabetic activity of the four dihydroxanthyletin-type coumarins . In addition, Pd-C-I, Pd-C-II, and Pd-C-III exhibited potent PTP1B inhibitory activity, with corresponding IC50 values of 4.32 ± 0.12, 6.17 ± 0.31, and 11.98 ± 0.43 µM, respectively. Similarly, as a reference compound, the α-glucosidase inhibition assay was validated with acarbose . A 5–15-fold increase in α-glucosidase inhibition compared to acarbose was observed in the coumarins studied. Among them, (+)-trans-decursidinol had a promising inhibitory effect of α-glucosidase with an IC50 value of 11.32 ± 0.56 µM, followed by Pd-C-I , Pd-C-II , and Pd-C-III . To determine the structure-activity relation between the dihydroxanthyletin-type coumarins and the target enzyme inhibition, their inhibitory effects on PTP1B and α-glucosidase were investigated. Therefore, given the coumarins’ PTP1B and α-glucosidase inhibitory activities, it can be speculated that the presence of a hydroxyl group at the 4′ position on the chroman ring plays an important role in the dihydroxanthyletin-type coumarins’ activity, and that substituting the hydroxyl group with other functional groups  decreased activity. The coumarins (+)-trans-decursidinol and Pd-C-I were more active than were the others, implying that the free hydroxyl group at the 4′ position has more influence on PTP1B and α-glucosidase inhibition than does the presence of acetyl or senecioyl groups at the 4′ positions of dihydroxanthyletin-type coumarins. Pd-C-II, which has a free hydroxyl group at the 3′ position, showed potent activities that inhibited PTP1B and α-glucosidase. Compared to our previous -1-naphthyl}-L-alanyl)amino] pentyl}oxy)-2-naphthoic acid (compound 23) was considered the standard ligand for validating the results of the simulation. The binding energies of dihydroxanthyletin-type coumarins with interacting residues, including H-bond and van derWaals residues, together with the number of H-bonds, are listed in trans-decursidinol formed five H-bonds with Ala27, Ile219, Gly220, Arg221, and Gly183, with a binding energy of –7.9 kcal/mol. In addition, hydrophobic interactions with Cys215, Gln266, Phe182, Thr263, and Ser216 residues stabilized the PTP1B-(+)-trans-decursidinol complex  -D: 3A4A) . Table 3residues B. Hydropresidues C. Pd-C-Iresidues D. As shotrans-decursidinol, followed by Pd-C-I, Pd-C-II, and Pd-C-III. The relative glucose uptake percentages of a 15 µM concentration of (+)-trans-decursidinol was 125%, and 40 µM concentrations of Pd-C-I, Pd-C-II, and Pd-C-III were 128%, 120%, and 117%, respectively, which were comparable to the positive control, metformin (relative % glucose uptake = 129% at 10 µM). Taken together, these results suggested that dihydroxanthyletin-type coumarins are able to induce insulin-resistant HepG2 cells’ sensitivity to insulin and increase the relative percentage of glucose uptake thereby.In order to investigate the capacity of coumarins’ for increasing glucose uptake, the MTT assay tested the cytotoxicity of four coumarins’ to HepG2 cells. A 2-[N- amino]-2-deoxyglucose (2-NBDG) test was used for testing of glucose uptake in insulin-resistant HepG2 cells. Treatment of high-dose of insulin-induced insulin resistance HepG2 cells as demonstrated by a substantial reduction in glucose uptake in the insulin-resistant group . Howevertrans-decursidinol diminished PTP1B’s expression at 10 and 15 µM, while Pd-C-I and Pd-C-II downregulated PTP1B’s expression at 20 and 40 µM. The order of the PTP1B inhibitory expression level was (+)-trans-decursidinol > Pd-C-I > Pd-C-II > Pd-C-III.Insulin signaling is negatively regulated by PTP1B and increased activity and expression are implicated in the insulin resistance pathogenesis. We have assessed its expression in insulin-resistant HepG2 cells by Western blot, to confirm that these coumarins improve insulin sensitivity by inhibiting expression of PTP1B. All dihydroxanthyletin-type coumarins inhibited PTP1B expression in a dose-dependent manner, which disclosed their insulin-sensitizing potential, as shown in —-induced nitration. As shown in —-mediated albumin nitration was significantly inhibited in a concentration-dependent manner by pretreatment with (+)-trans-decursidinol at various concentrations (5–40 µM). In addition, pretreatment at different concentrations (12.5–100 µM) with Pd-C-I, Pd-C-II, and Pd-C-III resulted in a pronounced dose-dependent inhibition of ONOO— -mediated albumin nitration  in order to determine if the anti-diabetic effects of dihydroxanthyletin-type coumarins’ could be due to a reduction oxidative stress. Exposure to t-BHP (200 μM) modifies the cells redox state, resulting in the formation of ROS. We observed that when the cells were treated with t-BHP (200 μM), ROS generation increased significantly to levels of approximately 100%, suggesting that t-BHP had a powerful effect on ROS generation in HepG2 cells. ROS production decreased in a co ncentration-dependent manner when the HepG2 cells were pretreated with (+)-trans-decursidinol, with 75.53%, 57.55%, and 43.44% inhibition at concentrations of 5, 10, and 15 μM, respectively -trans-decursidinol, Pd-C-I, Pd-C-II, and Pd-C-III exhibited strong ROS scavenging effects, with IC50 values of 9.77 ± 0.32, 6.32 ± 0.41, 10.76 ± 0.73, and 9.69 ± 0.21 µM, respectively, while the positive control, Trolox, had an IC50 value of 3.29 ± 0.26 µM.To evaluate the coumarins’ antioxidant effect, their potential to inhibit ROS and ONOO− was evaluated . The couDiabetes mellitus (DM) is a chronic metabolic disease that has a global effect on the health of humans. Hyperglycemia is the hallmark of diabetes attributable to either insulin resistance or insulin deficiency, or both . SeveralThe most important approach for T2DM treatment is optimum blood glucose levels after a meal. A more active approach to T2DM treatment has recently been involved in the disruption of dietary monosaccharide absorption by inhibiting α-glucosidase  and PTP1Another approach to treating diabetes is to delay the absorption of glucose by inhibiting an α-glucosidase carbohydrate-hydrolyzing enzyme, which is the main enzyme to catalysing the end-stage metabolism of carbohydrate, especially for non-insulin-dependent diabetes . The inhibitory action of this enzyme slows the production of D-glucose from dietary carbohydrates, delays the glucose uptake/reduces glucose levels and ultimately eliminates postprandial hyperglycemia. However, gastrointestinal side effects are associated with exiting clinical α-glucosidase inhibitors  . New phyA. decursiva L. is a well-known TCM herb of medicinal value for decades, and for medicinal purposes, the whole plant, leaves, and root parts of this plant were used. In our ongoing research into new diabetes phytochemicals, we have assessed the potential of the four major dihydroxanthyletin-type coumarins which are isolated from the whole plant [trans-decursidinol, Pd-C-I, Pd-C-II, and Pd-C-III were potent inhibitors of PTP1B. A. decursiva is a rich source in different types of coumarin derivatives, including dihydroxanthyletin, furanocoumarin, psoralen, pyranocoumarin, dihydropsoralen, hydroxycoumarin, and dihydropyran [trans-decursidinol, Pd-C-I, and Pd-C-II exhibited competitive-type PTP1B inhibition, while Pd-C-III showed mixed-type inhibition.le plant ,34 to indropyran . Among t23. In the corresponding ligand interactions of (+)-trans-decursidinol at PTP1B’s active site, there were five hydrogen-bonding interactions with five important residues  of the enzyme, and (+)-trans-decursidinol also bound at PTP1B’s catalytic site. Three major enzyme residues  participated in six hydrogen-bonding interactions in the case of Pd-C-I. In addition, five important enzyme residues  were involved in H-bonding interactions and residues, in which PTP1B’s catalytic site interacted with Pd-C-II. In addition, between the four coumarins, H-bonds were formed, and the main residues of Arg221 and Cys215 stabilized these coumarins and allowed them to fit together at the catalytic site of the enzyme. Shen et al. [Small molecule PTP1B inhibitors are difficult to detect since the PTP1B catalytic pocket is relatively shallow, minimizing the benefit of a small molecule inhibitor; however, the catalytic site consists of many polar amino acid residues  that selectively bind small molecules to single groups ,60. As wn et al. , who indtrans-decursidinol’s 4′-OH group formed a strong hydrogen bond with the Asp232 residue’s active site, while a ketone group and oxygen atom also exhibited two important hydrogen bonds with Trp229 and Ans496, and the Pd-C-I and Pd-C-II’s hydroxyl groups exhibited hydrogen interactions with the Asp232 and Gly408 residues. In addition, H-bonds between the four coumarins were formed and the main residues Trp229, Ans496, Lys506, Thr409, Arg412, Asp232, Ser497, and Ser505 stabilized these coumarins at the active site and allowed them to fit into the active pocket of the enzyme. Further, several hydrophobic interactions between the coumarins and active site residues were observed that stabilized their binding at the active site of α-glucosidase. Together, these observations indicate that these four coumarins do not bind to the same α-glucosidase catalytic site as acarbose.In addition, with AutoDock 4.2, a molecular docking analysis was performed to explore the binding mode of the coumarins’ inside the α-glucosidase binding pocket to understand their molecular interactions. For α-glucosidase, the docking results suggested that (+)-trans-decursidinol and Pd-C-I suppressed PTP1B expression in a concentration-dependent manner in Western blot study, while Pd-C-II and Pd-C-III significantly downregulated PTP1B’s expression. In the normal group, PTP1B was expressed weakly, but in the insulin-resistant group, it intensified. While PTP1B inhibition is known to boost insulin sensitivity effectively, there is still a lack of a better understanding of the regulatory mechanism of PTP1B. Some PTP1B inhibitors have been found to inhibit IR and IRS phosphorylation, contribute to insulin resistant, and activates insulin signaling by overexpression of PTP1B [In order to further explore coumarin’s diabetic mechanisms, we have studied the impact of these compounds on the insulin sensitivity in normal versus insulin resistant HepG2 cells. PTP1B is a well-known negative insulin-signalization pathway regulator . An indiof PTP1B ,61,62,63t-BHP in HepG2 cells. ONOO− is a highly reactive oxidizing and nitrating agent derived from NO. and ·O2- that oxidizes cellular components, such as carbohydrates, proteins, DNA, and lipids [− [− and inhibited ONOO−-mediated tyrosine nitration significantly in a dose-dependent manner. These findings showed that the anti-DM properties of coumarins can be due to the direct hepatocytes scavenging of ONOO− and total ROS.ROS and reactive nitrogen species (RNS) are considered to play a central role in DM- associated liver diseases. Therefore, to avoid or alleviate liver diseases associated with oxidative stress, the use of antioxidants has been proposed. It is believed that the antioxidant effects of phenolics, particularly polyphenols, are due at least in part, to the chelation of metal ions, the prevention of radical formation, the indirect modulation of enzyme activity, and modulations of antioxidant and detoxifying enzymes expression ,65. Reced lipids , and canipids [− . Both coA. decursiva-derived dihydroxanthyletin-type coumarins. The evidences showed that coumarins enhanced insulin-sensitivity by downregulating PTP1B and promoted the uptake of glucose in insulin-resistant HepG2 cells. The coumarins also appeared to exert their anti-DM effects by scavenging ONOO− and suppressing ONOO−-mediated albumin nitration effectively by reducing total and intracellular ROS production. Therefore, A. decursiva-derived dihydroxanthyletin-type coumarins could also supplement pharmacotherapy in the treatment of diabetes. Further, in vivo studies are also important for validating our findings.In conclusion, the study indicates the ability to treat DM therapeutically with PTP1B and α-glucosidase inhibited by


Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe, and humans construct the ring structure with one, two, and three myosin-II isoforms, respectively. Here, we provide a comparative analysis of the roles of the non-muscle myosin-IIs in cytokinesis in these three model systems, with the goal of defining the common and unique features and highlighting the major questions regarding this family of proteins.The contractile ring, which plays critical roles in cytokinesis in fungal and animal cells, has fascinated biologists for decades. However, the basic question of how the non-muscle myosin-II and actin filaments are assembled into a ring structure to drive cytokinesis remains poorly understood. It is even more mysterious why and how the budding yeast  Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe, and humans possess one, two, and three isoforms of the NM-IIs, respectively  play critical roles in many fundamental processes including cytokinesis, cell adhesion, cell migration, exocytosis, and tissue morphogenesis . Interesectively . While tensively , the in MYO1) in budding yeast, two (MYO2 and MYP2) in fission yeast, and three  in mammalian cells. Here, we attempt to shed some light on this mystery by comparative analysis of the roles of the NM-IIs in cytokinesis in these diverse model systems.Cytokinesis in fungal and animal cells requires concerted actions of an actomyosin ring (AMR), targeted vesicle fusion, and localized ECM remodeling . The AMRS. cerevisiae has only one myosin-II heavy chain Myo1   , one ess reason) . Mlc1 is reason) . Deletiokgrounds . Thus, tin vitro without the assistance of some accessary factors.Myo1 consists of 1,928 amino acids, with a predicted globular head (aa1–855) containing an actin-binding site and ATPase domain, two IQ motifs (aa795–850) where the ELC and RLC bind, and a coiled-coil (CC) tail  . RemarkaMyo1 localizes to the division site in a biphasic pattern . Before Because Mlc1, the ELC for Myo1 , is alsoHOF1, which encodes an F-BAR protein involved in cytokinesis, the C-terminal region of Myo1 must be important for its function (Fluorescence recovery after photo-bleaching (FRAP) analysis indicates that prior to anaphase, there is little or no exchange of Myo1 between the division site and the cytosol, suggesting that nearly all the Myo1 molecules are localized to the division site. However, Myo1 is highly dynamic within the division site . This dyfunction .Myo1 localizes to the presumptive bud site and then to the bud neck from bud emergence to the completion of cytokinesis . However, actin filaments join Myo1 at the bud neck to form an AMR only after the onset of anaphase. Thus, the function of Myo1 during the cell cycle can be divided into two stages: before and after anaphase.Myo1 facilitates the retrograde flow of actin cables before anaphase . RetrogrIn budding yeast, AMR constriction is closely followed by the centripetal growth of a primary septum (PS) that is catalyzed by the chitin synthase II (Chs2) . These tin vitro and whether its filament assembly in vivo is regulated by cell cycle-controlled phosphorylation and/or by trans-acting factors such as other cytokinetic proteins. Finally, it remains unclear how Myo1 is spatiotemporally coupled to polarized exocytosis and Chs2-mediated PS formation during cytokinesis.FRAP analysis suggests that Myo1 is organized into a stable structure during cytokinesis . Indeed,S. pombe, evolutionally diverged from its fungal relative, the budding yeast S. cerevisiae, ∼420 million years ago   . Howeverich NHR) . Moreoveich NHR) . It is tokinesis .The assemblies and spatial organization patterns of Myo2 and Myp2 within the contractile ring are distinct . There iWhile the assembly of Myo2 in the cytokinetic nodes or ring is best understood among all the NM-IIs, the assembly of Myp2 is far from clear. Disruption of F-actin by Latrunculin A (LatA) abolishes Myp2, but not Myo2, localization, suggesting that Myp2 localizes to and maintains at the division site by binding to actin filaments or other proteins whose localizations depend on F-actin . This isS. pombe localize to the division site during mitosis but at distinct times  in the myo2-E1 allele induces a steric hindrance that causes deficiencies in ATPase activity and actin-filament binding in vitro (myo2-E1 cells can grow and divide reasonably well at the permissive temperature whereas myo2 null is inviable suggests that a motor-independent function of Myo2 likely exists. A genetic screen for the suppressors of the temperature-sensitive growth of the myo2-E1 cells has led to the identification of an intragenic suppressor (Sup1) that contains two amino-acid changes, Q640H and F641I , Bgs1 localizes to the misoriented myosin-II cables, instead of a ring structure, at the medial region and starts synthesizing the PS materials by following the myosin cables  can largely accomplish cytokinesis and guide PS formation   . Further mutant) , similar mutant) . Taken tF-actin is also involved in guiding the PS formation, but is only required during the early phase of ring constriction. Association of Bgs1 with the myosin-II and the activation of PS formation can occur in a F-actin-independent manner . Howevermyp2+, but not the myo2-E1 mutant, leads to loss of viability and defects in cytokinesis in the presence of high salt  encoded by MYL9, MYL12A, or MYL12B  to the diameter of the intercellular bridge (ICB) between the daughter cells at the time of the midbody formation (1.5–2 μm) , 1977. TDuring furrow ingression, all three isoforms localize to the division site and play redundant or overlapping roles, as depletion of IIA or IIB alone or in combination did not block furrowing in HeLa-Kyoto cells, but the addition of 25 μM blebbistatin, a specific inhibitor of myosin-IIs , completIt has become increasingly clear that there are two spatiotemporally separated actomyosin rings that operate during mammalian cytokinesis . The priSeveral recent studies indicate that IIA generates cortical tension and drives furrow ingression during cytokinesis, whereas IIB controls cortical stability and cytokinetic fidelity, perhaps by attenuating the IIA-dependent rate of rapid ingression . These oin vitro behaviors such as in motility and filament assembly, their mechanisms of action for their in vivo roles such as in cytokinesis remain poorly understood. There are many outstanding questions. For example, it remains unknown how and why different isoforms are regulated to achieve a cell type-specific ratio to drive furrow ingression and abscission. What is the architecture of the primary and second-stage AMRs? Is the myosin-II organization in the contractile ring nearly identical to that in stress fibers, as suggested by a recent analysis of IIA at the division site in HeLa cells  to the onset of anaphase . This poSecond, it is remarkable that, in all three systems, cytokinesis can largely occur in the absence of the motor activity of the NM-IIs or when the motor activity is severely compromised, but the execution of cytokinesis requires the binding of the NM-IIs to the actin filaments. This is demonstrated abundantly clear by the behaviors of the headless AMR in budding yeast , and of Finally, why do the budding yeast, fission yeast, and mammalian cells possess one, two, and three NM-II isoforms, respectively? There are a number of possibilities. First, it may be related to the dimension of the division site in different systems . The divKW and HO wrote the initial draft. EB revised the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.