Cordilleran orogenic systems have complex, polycyclic magmatic and deformation histories, and the timescales and mechanisms of episodic orogenesis are still debated. Here, we show that detrital zircons (DZs) in terrigenous sediment from the late Pleistocene Amazon Fan, found at the terminus of the continent-scale Amazon River-fan system, record multiple, distinct modes of U-Pb crystallization ages and U-Th/He (ZHe) cooling ages that correlate to known South American magmatic and tectonic events. The youngest ZHe ages delineate two recent phases of Andean orogenesis; one in the Late Cretaceous – Paleogene, and another in the Miocene. Frequency analyses of the deep-time Phanerozoic record of DZ U-Pb and ZHe ages demonstrate a strong 72 Myr period in magmatic events, and 92 Myr and 57 Myr periods in crustal cooling. We interpret these results as evidence of changes in upper and lower plate coupling, associated with multiple episodes of magmatism and crustal deformation along the subduction-dominated western margin of South America. South American cordilleran orogenic systems have repeated complex magmatic and deformation histories. Here the authors analyze detrital zircons found in the Amazon deep-sea fan that record mountain-building events and reveal cycles of orogenesis with periods of ~60–90 Myr since the Phanerozoic. Conceptual models link episodic magmatism in cordilleran arc systems to a suite of related processes in the mantle and upper plate, including tectonic activity in the orogen, and resulting surface processes . For example, one version of the orogenic cycle consists of periods of high magmatic flux associated with underthrusting of melt-fertile lower crust, delamination of a dense mafic orogenic root from the upper plate, and subsequent rapid isostatic uplift, tectonic growth of the fold-thrust belt, and increased orogen relief associated with enhanced erosion rates . Datasets of multiple thermochronometers from clastic detritus have previously been used to propose anti-correlation between the timing of magmatic flux events, and tectonic denudation of an orogen (Carrapa and DeCelles4). And while orogenic cycles are proposed to occur at quasi-periodic timescales of ~25–50 Myr , or ~70 Myr , the nature and timing of erosional response to orogenic cycles are still unclear.Globally, cordilleran systems with volcanic arcs exhibit episodic cycles of magmatism, which are often constrained by the measurement of U-Pb ages of zircons in igneous and detrital material (Paterson and Ducea6), and, in some cases, ultimately transferred to passive margin marine stratigraphic archives . Deep-sea fans—large accumulations dominated by terrigenous sediment—record the physical and chemical signatures of their linked continent-scale river catchments (Hessler and Fildani9). The Amazon Fan, offshore NE South America . Since this terrigenous detritus records the tectonic history of the catchment, it is useful in the recovery of past thermal and deformation events in the cordilleran system. The application of low-temperature detrital thermochronometry could help clarify the tempo of magmatic and thermal events experienced by South America’s active western margin over numerous timescales.Thermal events in the orogenic belt are preserved in the detritus shed from cordilleran systems into their associated basins . We perform frequency analyses of U-Pb and ZHe age data, and present the detrital geochronologic and frequency analyses results in the context of: (1) provenance interpretations of Amazon Fan sediment, and (2) past orogenic events of the South American continent, including generation of zircons during phases of magmatism, metamorphism, and their low-temperature cooling histories associated with the exhumation of the Andes and predecessor orogenic systems.Here, we characterize the thermotectonic histories experienced by detrital zircons (DZs) found in the late Pleistocene Amazon Fan through the application of combined DZ U-Pb and U-Th/He double dating .Western South America is dominated by the high-elevation, cordilleran system formed through the subduction of oceanic lithosphere, arc volcanism, and deformation of the overlying South American plate. The orogenic system is divided, from west to east, into a forearc region consisting of a western cordillera containing the magmatic arc, and a retroarc region, consisting of an eastern cordillera, in the case of the central Andes a broad high-elevation plateau, and an active retroarc fold thrust belt (Horton12). During the Paleogene, rates of shortening in the central Andes increased dramatically , as did the elevation of the central Andes  culminating in the Miocene with rise of the central Andean Plateau . Andean retroarc foreland basin fill records increased accommodation and initiation of widespread coarse grained sedimentation starting in the Late Cretaceous to early Paleogene, and is interpreted to be the signal of shortening induced lithospheric flexure (Horton11 and references therein). A geographically widespread hiatus or cessation of coarse grained sedimentation occurred across the Eocene, with renewed coarse grained sedimentation recorded in retroarc foreland basins starting in the Oligocene (Horton11)The recent phase of Andean orogenesis initiated due to subduction of the Nazca plate beneath the South America plate during the late Mesozoic, and may have progressed in time from the northern Andes toward the central and southern Andes . Early orogenic events experienced by western South America are the Neoproterozoic through Paleozoic assembly of Gondwana, including the Pan African-Brasiliano (Neoproterozoic), the Pampean (~Cambrian) and the Famatinian (~Ordovician) orogenies, respectively . These widespread events were followed by phases of Permian-Triassic metamorphism , and Jurassic magmatism along the western margin of South America . During the Mesozoic, prior to widespread Andean shortening, extension and post-rift thermal subsidence were the dominant modes of lithospheric deformation along much of what is now the central Andes (Horton14), leading to the formation of large sedimentary basins along strike in the Cretaceous (Horton11).Subduction related deformation along the pre-Andean margin was polycyclic, long lived and regional in extent (Chew21). Northern Andean streams with high elevation headwaters in southern Colombia and Ecuador tap into an active volcanic arc, Paleogene and Cretaceous sedimentary units, Neoproterozoic metamorphic units, Triassic metamorphic, and Jurassic volcanic and plutonic units in the retro arc fold-thrust belt .Andean streams are tributaries to the Amazon River, which transfers clastic detritus from a wide array of lithologies, representing a large span of geologic time, depositional environments, and igneous and metamorphic facies . There, Andean streams in Peru tap Mesozoic and Paleozoic sedimentary, volcanic, and metamorphic units in the eastern cordillera, and Cenozoic, Cretaceous, and Jurassic sedimentary units in the fold-thrust belt . South of ~15° S, in the western cordillera of Peru, lies an active volcanic arc. However, most volcanic detritus is transferred to internally drained, high elevation basins within the Altiplano of Peru and Bolivia. The eastern cordillera, and fold-thrust belt of southern Peru and Bolivia are dominated by Cenozoic, late Mesozoic, and extensive Paleozoic sedimentary units .A magmatic gap related to Peruvian flat slab subduction exists between ~2° and 15° S . Consequently, DZ age spectra of sediment from modern rivers of the Amazon system display evidence for recycling of zircons originally sourced from the craton . Specifically, modern river sediments from the Andes may contain DZs with age components related to the Meso-Neoproterozoic Sunsás orogen , the Neoproterozoic to Cambro-Ordovician Famatinian—Pampean orogens, and U-Pb ages related to periods of Mesozoic through Cenozoic magmatic arc activity along western South America . DZs with U-Pb ages >1300 Ma correspond to cratonic terranes and are documented in modern Andean sourced rivers and in bedrock sources within the northern to central Andes . Pampean aged DZs are sometimes referred to as Pan African—Brasiliano, an orogeny that affected eastern South America, and which can also be found in modern Andean streams. Due to long term sediment recycling in the Andes and sedimentary basins of the fold-thrust belt, the provenance of DZs in the lower Amazon River and Fan becomes difficult to discern . We address this issue with the application of double dating of DZs, where recent tectonic exhumation may be revealed by the low-temperature cooling history of cratonic aged DZs sourced from the Andes.The modern east-flowing Amazon River is a relatively recent feature. An ancestral Amazon River once flowed east to west, supplying sediment from the craton to Andean retroarc foreland basins, until a phased reversal was completed in the Miocene, as marked by the transfer of Andean detritus to the Atlantic Ocean , yet contribute a disproportionate amount of sediment to the Amazon fluvial system and related deep-sea fan . Direct observations of river suspended sediment loads , mud geochemistry from the Amazon Fan , and DZ provenance estimates along the lower Amazon River and deep-sea Fan suggest that a large proportion of sediment (~80–90%) may be sourced from the Andes . Differential zircon fertility of source terranes has been shown to influence DZ age distributions in some cases (Moecher and Samson33), which may interfere with provenance interpretations. However, denudation rates of Andean streams that are an order of magnitude higher than low-lying Amazonian streams are likely a dominant process driving provenance signatures in the Amazon Fan. Given the source of sediment in the Amazon Fan is largely from the cordilleran system, the Amazon Fan sedimentary record should preserve the long thermal history of the tectonically active western margin of South America.Onshore, catchments within the Amazon Basin draining high relief mountainous areas represent a small fraction of the total Amazon drainage basin area . These first cycle volcanic DZs range in age from ~30 Ma to 585 Ma, with the majority (four of six) from Mesozoic and Cenozoic U-Pb age groups . The remaining three Cenozoic DZs have ZHe ages that are several Myr younger (~3–10 Myr) than their associated U-Pb ages.Six first cycle volcanic DZs were identified by their overlapping U-Pb and ZHe ages  are Cenozoic, 22% (n = 25/112) are Mesozoic, 29% (n = 32/112) are Paleozoic, and 12% (n = 13/112) are Precambrian in age , yet only two ZHe ages record cooling associated with that period of time (900–1300 Ma). Instead, the Sunsás U-Pb age group is associated with ZHe cooling ages between ~200 and 600 Ma, and a distinct mode of Late Cretaceous through Neogene ZHe ages, , rather than the more areally extensive but low relief Amazon craton. Indeed, while the Andes represent a relatively small fraction  of the Amazon drainage basin (~12%), the Cordilleran system contributes a disproportionate amount of DZs with Cenozoic ZHe cooling ages  to the Amazon Fan. DZs with U-Pb ages <400 Ma are likely sourced entirely from the Andes  are scarce (n = 4/112), ZHe cooling ages display the most pronounced mode between 0 and 50 Ma .The distribution of DZ U-Pb and U-Th/He double dates from sediment of the Amazon Fan is representative of clastic detritus transferred from the Amazon River to the Atlantic Ocean over late Pleistocene timescales Figs.  and 2. Ades Fig. , as most36). Although Neoproterozoic through craton aged DZs are documented in Andean rivers , Cenozoic cooling ages associated with those U-Pb age groups provides another line of evidence that recycling  of older cratonic DZs is common in the Cordillera of South America.Craton aged DZs (>1300 Ma) correspond largely to Neoproterozoic and Paleozoic ZHe cooling ages, which could be interpreted as sediment recycling in the cordillera or primary cratonic sources. Exhumation between 400 and 500 Ma involved uplift and erosion of Sunsás (900–1300 Ma) through early Paleozoic aged rocks, as shown by the U-Pb and ZHe relationships of DZs Figs. . The mod2 explicitly predicts an association between high magmatic flux events and creation of orogenic relief, driven by underthrusting of melt fertile crust, and isostatic surface uplift in response to delamination of dense lithospheric mantle beneath the orogen. Before or during such a phase of high magmatic flux, the width of the orogen is thought to grow as the fold-thrust belt propagates, resulting in exhumation and cooling of crustal material. In our dataset, distinct modes of ZHe cooling ages partially overlap with, precede, or temporally lag modes of U-Pb ages, suggesting a relationship between magmatic flux events (modes of U-Pb ages), and enhanced crustal cooling . Previous reports of timescales for cyclicity were focused on relatively specific and restricted geographic extents during the Mesozoic through Cenozoic. The Amazon Fan samples both the north central and northern Andes, with a ZHe record that spans the Phanerozoic. This broad geographic and temporal sampling may explain moderate differences in reported periodicity across multiple studies, especially given the more recent record of diachronous mountain building events along strike in the central to northern Andes .The periods of orogenic activity, as archived in the Amazon fan, are reasonably consistent with prior estimates of cyclicity in the central to northern Andes , to the Andean retroarc foreland from Columbia, Central Peru, and southern Bolivia .Figure 3; DeCelles et al.2; Sundell et al.5). For example, Jurassic through Neogene orogenesis is well documented during the formation of the modern Andes ; ZHe ages from the Amazon Fan record phases of magmatism and increased crustal cooling during the Jurassic (~180 Ma), Cretaceous (~110 Ma) and latest Cretaceous – early Paleogene (~60–70 Ma), and early late Miocene (~12 Ma) , for example in the extensive Paleozoic metamorphic and siliciclastic units exposed in the eastern Andes of Bolivia and Peru . This would explain the abundance of Paleozoic ZHe cooling ages found in what is hypothesized to be largely Andean derived clastic sediment from the northern central and northern Andes.We hypothesize numerous exhumational cooling events remain recorded in un-reset ZHe cooling ages from upland Andean bedrock areas of the eastern Cordillera , double dating of DZs significantly improves the interpretations of provenance, and more so, the geologically recent thermotectonic histories experienced by DZs .DZ U-Pb ages are rarely reset during multiple phases of orogenesis, hindering interpretations of geologically recent and shallow exhumation histories (<5 km). Although quantitative mixture models applied to DZ age spectra have improved sediment provenance interpretations in the Amazon and other systems , zircons do not retain measurable amounts of 4He; once a zircon is advected through its closure temperature range , the zircon will begin to retain 4He in measurable amounts that are a function of He diffusion kinetics and time since passage through the closure temperature. While U-Pb ages record primary crystallization from magmatic sources, U-Th/He dating of individual zircons yields information about the most recent exhumation history experienced by a zircon.ZHe thermochronology exploits the thermal diffusivity of 7). DZs for double dating were further screened based on grain size, and morphology suitable for the ZHe technique . DZs were selected from five samples of turbiditic sands collected from two sediment cores recovered during Ocean Drilling Program Leg 155 . We amalgamate these subsamples .We measured 114 ZHe cooling ages in previously dated (U-Pb) DZs from the Amazon Fan Fig. . Selecti2; Sundell et al.5) we analyzed the KDE interval between 650 and 40 Ma of the U-Pb and ZHe record . To estimate the spectral characteristics of the record we used the R package Astrochron (Meyers52) to carry out power spectra analysis using the Mann and Lees53 robust red noise Multi-Taper method (MTM) analysis to test for statistical significance of spectral peaks. Prior to analysis the KDE was interpolated to even sample spacing, prewritten using an autoregressive-1 (AR1) filter, and detrended. We report periodicities that exceed the 85% and 90% MTM harmonic F-test confidence level estimate, while also achieving the required robust red noise confidence level (85%) within ± half the power spectrum bandwidth resolution. This method of estimation substantially reduces the identification of false positive estimates of spectra peaks (Meyers54). The results of the frequency analyses (Fig. In order to quantitatively evaluate the periodicity of hypothesized cyclic orogenic processes (e.g., DeCelles et al.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1


Cache Valley virus (CVV) is a mosquito-borne bunyavirus that is enzootic throughout the new world. Although CVV is known as an important agricultural pathogen, primarily associated with embryonic lethality and abortions in ruminants, it has recently been recognized for its expansion as a zoonotic pathogen. With the increased emergence of bunyaviruses with human and veterinary importance, there have been significant efforts dedicated to the development of bunyavirus vaccines. In this study, the immunogenicity of a candidate live-attenuated vaccine (LAV) for CVV, which contains the deletion of the nonstructural small (NSs) and nonstructural medium (NSm) genes (2delCVV), was evaluated and compared with an autogenous candidate vaccine created through the inactivation of CVV using binary ethylenimine (BEI) with an aluminum hydroxide adjuvant (BEI-CVV) in sheep. Both 2delCVV and BEI-CVV produced a neutralizing antibody response that exceeds the correlate of protection, that is, plaque reduction neutralization test titer >10. However, on day 63 postinitial immunization, 2delCVV was more immunogenic than BEI-CVV. These results warrant further development of 2delCVV as a candidate LAV and demonstrate that the double deletion of the NSs and NSm genes can be applied to the development of vaccines and as a common attenuation strategy for orthobunyaviruses. Peribunyaviridae, order Bunyavirales). CVV was first isolated in Cache Valley, Utah, USA, in 1956 from Culiseta inornata  is an emerging zoonotic mosquito-borne orthobunyavirus , medium (M), and large (L), which code for various structural and nonstructural (NS) proteins , Bunyamwera virus (BUNV), Schmallenberg virus (SBV), and La Crosse virus , a phlebovirus, can lead to lower infection and dissemination rates in mosquitoes compared with the wild type (wt) virus  LAV. The immunogenicity of the 2delCVV candidate vaccine was evaluated in sheep and then compared with the autogenous vaccine generated by the inactivation of CVV with binary ethylenimine (BEI-CVV) based on serum neutralizing titers in sheep.l-glutamine (Thermo Fisher Scientific) as previously described  supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), 10% tryptose phosphate broth , penicillin/streptomycin (Thermo Fisher Scientific), and Cs. inornata in Cache Valley, Utah, in 1956 /mL was inactivated, and the complete inactivation was confirmed by the lack of infectivity detected by plaque assay  conditions. Animals were allowed an acclimation period of 5 days in the BSL2-Ag housing before the start of the experiments. Throughout the experiment, all animals were given n = 10), the BEI-CVV group (n = 10), or the L-15 media group (n = 4).The objective of this experiment was to evaluate and compare the immunogenicity of a candidate LAV for CVV and an autogenous inactivated vaccine for CVV. After the primary immunization, boosters on days 21 and 42 were used to determine whether either vaccine increased the titers of neutralizing antibodies that would likely provide a protective immune response. Twenty-four 6-month-old male Rambouillet lambs were first assigned to one of the following vaccine groups: the 2delCVV vaccine group (50). On day 0 of the experiment, lambs were immunized subcutaneously at their fore flank (right behind their elbow) with their corresponding immunization regimen, including (1) 1 mL 2delCVV containing 105 pfu of infectious viruses, (2) 1 mL BEI-CVV with the addition of the aluminum hydroxide adjuvant, or (3) 1 mL of L-15 media.Before initial immunization, all animals were determined to be healthy and seronegative to CVV through the analysis of collected serum using 50% plaque reduction neutralization tests , depression, weight loss, respiratory distress, lameness, neurological signs, and vaccine site reactions. 50 was performed as previously described  was used to determine the significant difference among and between the candidate vaccine's PRNT50 titers from the initial and booster vaccinations. To evaluate the significant difference between the two vaccine groups over the duration of the study, a two-way ANOVA was performed.All statistical analyses were conducted using the GraphPad Prism (version 8.1.2) program . The Shapiro–Wilk test was used to test for normal distribution. PRNTThe animals were healthy and seronegative to CVV before immunization. Animals from both the live attenuated candidate 2delCVV vaccine group and the inactivated BEI-CVV vaccine group presented no observable adverse clinical signs, weight loss, or increased rectal temperatures, however, all animals that received BEI-CVV had swelling averaging 3 cm in diameter present at the injection site after vaccination that persisted for the duration of the study. Animals were bled on days 3, 5, 7, and 14 to determine immune responses after the primary immunization, and on days 20, 35, 41, 56, and 63 to determine whether booster immunizations would increase the immunogenicity and produce a long-lasting neutralizing antibody response with each candidate vaccine.50 titer, that is, immune correlate of protection, after a single immunization on day 20 postinitial immunization. There was no significant difference in the neutralizing antibodies elicited by both vaccines when compared  receiving BEI-CVV waned to 5 and 10, whereas all animals receiving 2delCVV maintained robust neutralizing activity with all animals ending the study with PRNT50 titers of 160.Importantly, immunization of BEI-CVV only led to transient neutralizing antibody responses. The serum PRNTIn this study, we evaluated the immunogenicity of the candidate 2delCVV LAV and an autogenous CVV vaccine, BEI-CVV. Although both vaccines provided a neutralizing antibody response past the threshold of immune protection, animals immunized with 2delCVV LAV developed higher serum neutralizing titers at 63 days postimmunization. We conclude that 2delCVV is superior in immunogenicity and can be further evaluated as a candidate veterinary LAV.Inactivated vaccines are generally safe and can be developed within a relatively short period of time, making them a putative counter measure against abrupt outbreaks of viral diseases. The inactivated candidate vaccine used in this study was created similarly to one of the inactivated vaccine candidates used against SBV (Wernike et al, However, as expected, the immunogenicity of the inactivated vaccine is low and leads to the requirement of at least a two-dose immunization regimen to produce sufficiently high serum neutralizing antibodies, the correlate of protection. This indicates that the first dose may only give partial protection and the maximum immune response is not achieved until after one or two booster immunizations. This justifies the development of 2delCVV as a candidate LAV. This study proved the superior immunogenicity of 2delCVV compared with CVV-BEI, suggesting that a similar approach to rationally design candidate LAVs may offer more effective tools for the control of CVV and other related orthobunyaviruses.A challenge study would be necessary to determine actual protection in pregnant sheep against the fetal malformations and abortions that infection with CVV causes. Such challenges could not be performed during the current studies. Since CVV does not normally display clinical symptoms in adult ruminants, challenging the sheep used in this study would not have given relevant information on protection. Future studies could include a challenge study conducted to examine safety and protection in pregnant ruminants, evaluating the cytokine profile, and determining the vaccine-induced Immunoglobulin G/Immunoglobulin M host antibody response by antigen-specific indirect enzyme-linked immunosorbent assays.Previously an inactivated vaccine for RVFV reduced viremia with a lack of clinical signs in a vaccinated lamb, however, there were no detectable neutralizing antibodies (Kortekaas et al, LAVs are typically considered to be easier to produce and more efficacious. Modified live vaccines were initially based on random introduced mutations through serial passages in cell culture or in the presence of chemical mutagens (Henderson, The live attenuated candidate 2delCVV vaccine was created with the inability of the virus to express the NSs and NSm genes. This attenuated the virus so it would elicit an immune response without the ability to revert to full virulence. The complete deletion of genes could possibly enable us to distinguish vaccinated from field-infected animals. This could potentially be achieved through the detection of the wt virus using an NSs or NSm antibody and through detection of the mutant virus using an anti-NSs or anti-NSm antibody as previously described (Bird et al, Although experiments with BUNV have demonstrated how NSs was nonessential in mosquito cell lines, NSs was shown to be important for the infection of mosquitoes (Szemiel et al, As expected, the autogenous BEI-CVV vaccine was capable of eliciting a neutralizing antibody response, however, a booster immunization would be needed and there was an adverse reaction at the injection site. The 2delCVV candidate vaccine not only produced a neutralizing antibody response through the duration of the study that could confer protection, but it also produced a more robust neutralizing antibody response at the end of the study when compared with the autogenous vaccine. Therefore, through the deletion of the NSs and NSm genes, an immunogenic vaccine for CVV was developed. Although protection studies are warranted, these data provide a basis for further development of immunogenic vaccines for CVV and other related orthobunyaviruses.


Arabidopsis  is an ideal model plant for studying the impact of environmental stress on plants’ growth and development because the ways in which Arabidopsis adapt to these stresses resemble those of many plants, and therefore, conclusions obtained from these scientific studies have often been used as the universal reference for other plants. This study reviewed the main findings of present-day interactions between MNPs and Arabidopsis thaliana from plant internalization to phytotoxic effects to reveal the mechanisms by which nanomaterials affect plant growth and development. We also analyzed the remaining unsolved problems in this field and provide a perspective for future research directions.The widespread application of metal-based nanoparticles (MNPs) has prompted great interest in nano-biosafety. Consequently, as more and more MNPs are released into the environment and eventually sink into the soil, plants, as an essential component of the ecosystem, are at greater risk of exposure and response to these MNPs. Therefore, to understand the potential impact of nanoparticles on the environment, their effects should be thoroughly investigated.  Metal nanoparticles (MNPs) are widely used in biosensors, medical imaging, diagnostic and therapeutic materials, antimicrobial agents and drugs, chemical catalysis, optoelectronics and other areas due to their unique physical and excellent chemical properties ,3,4,5,6.2 NPs were found to promote seed germination in tomato, onion and radish, Cu NPs concentration-dependently inhibit seedling growth and root growth in both mung bean and wheat, and Al2O3 NPs were observed to have no pharmacological effect on the root elongation of Arabidopsis, radish, rape, ryegrass, lettuce or cucumber . T. TArabidArabidopsis while positively charged nanoparticles uptake occurred to a small extent, possibly through other processes, such as clathrin-mediated endocytosis, in the phytoplankton  as Zn fertilizer has been shown to be effective for correcting Zn deficiency in soils . Howeverransport ,35. Prak element  36]..ArabidopArabidopsis was exposed to 5–40 mg/L of CuO NPs, the Cu content in Arabidopsis roots was significantly increased compared to the Cu content in Arabidopsis stems and leaves. Additionally, while the transfer rate of CuO NPs from root to shoot was found to be low (1.1–2.8%), under the same conditions, that of Cu2+ occurred at a higher rate (10.8%), indicating a weak transport capacity of CuO NPs  could be absorbed, translocated and distributed among the tissues and cells of Arabidopsis seedlings [2 NPs, García-Sánchez et al. observed that TiO2 NPs could enter Arabidopsis cells, accumulate in subcellular (including vesicular) locations such as the cytosol and root cell nuclei and further disrupt Arabidopsis microtubule dynamics [Arabidopsis, and it would be helpful to further assess TiO2 NPs using traceable signals.Although Ti elements are non-essential elements for eedlings . Via mordynamics ,40,41. T2 NPs are a class of MNPs that tend to aggregate and precipitate in aqueous solutions due to their size and surface properties. In a study by Yang et al., the investigators introduced an agar curing medium to prevent the aggregation of CeO2 NPs, allowing them to be uniformly dispersed. It was found that the transport of Ce compounds by Arabidopsis grown in the agar medium behaved similarly to internalized CuO NPs in plants [Arabidopsis exposed at 0–1000 ppm CeO2 NPs by ICP-MS and observed measurable amounts of the elements in the root and stem tissues of Arabidopsis. However, the underlying mechanism of this transport is yet to be uncovered. Despite these observations, the accumulation and translocation of CeO2 NPs in plants seem to vary depending on the plant species. Birbaum et al. found that CeO2 NPs did not undergo translocation in maize, while Ce elements were found to be accumulated in plants such as alfalfa, cucumber and tomato [CeOn plants . Ma et ad tomato ,44,45,46Arabidopsis. In addition to the barrier created by the plant’s cell wall, when QDs are electrostatically adsorbed on the root surface, they form bulky agglomerates, which further impedes their entry as endocytosis cannot occur [Given their promising water solubility and small size, quantum dots (QDs) were believed to be easily absorbed by plants; this was also confirmed in the recent study of pumpkin’s physiological responses to zinc oxide quantum dots and nanoparticles . The expot occur .Arabidopsis root cell membranes, they have a low rate of internalization [Arabidopsis. Besides this, the importance of nanomaterials’ entry through stomata has also been extensively studied [Taken together, the current body of literature suggests that although the uptake of most MNPs is associated with ion transporters on lization . Small n studied ,52. MoreArabidopsis, most studies confirmed that MNPs have predominantly harmful effects on the plant. Arabidopsis.The large surface area and small size of NPs are some of their desirable attributes that allow them to substantially ameliorate plants’ physiological processes. Nevertheless, the results derived from such research have not always been positive as NPs have been shown, in some cases, to negatively affect plants due to their potentially toxic nature . DespiteArabidopsis, while Ag NPs at concentrations of 50 mg/L and 60 mg/L had a positive effect on root growth. In addition, the shapes of Ag NPs were also found to be an important factor influencing root growth [Arabidopsis seedlings [Growth potential, seed germination, biomass and leaf surface area are the commonly used parameters to assess morphological changes in plants and phytotoxicity ,76,77,78t growth . Ag NPs eedlings .Arabidopsis lateral roots have been found to be significantly reduced after treatment with different sizes and concentrations of Au NPs solutions, whereby smaller (10 nm) Au NPs negatively affected primary root growth but significantly promoted root hair growth  are believed to interact with ed yield . These rArabidopsis may also be related to the alteration of the porosity of the plant’s cell walls when stimulated under different concentrations of MNPs. While larger pores tend to promote the uptake of nutrients and MNPs from the soil, MNPs tend to simultaneously cause root clogging and impede the entry of nutrients.The morphotropic effects of MNPs on 2+ is the primary mechanism by which CuO NPs induce their phytotoxic effects [2+ contributed only a tiny fraction to the toxicity induced by CuO NPs. Upon 2 h of exposure to CuO NPs (10 mg/L and 20 mg/L), severe damage of Arabidopsis root cells was observed, whereas corresponding exposure with dissolved Cu2+ (0.80 mg/L and 1.35 mg/L) did not damage the roots and only exhibited partial phytotoxic effects after 12 days of exposure [Arabidopsis roots, leaves, flowers and harvested seeds treated with CuO NPs contained significantly higher Cu elements than CuO bulk particles (BPs) and Cu2+ treatment, demonstrating that CuO BPs cannot be readily accumulated and distributed in the plant , they induce glutathione metabolism (oxidative stress response) and inhibit chlorophyll production and plant growth [4+, and are particle-specific [At low concentrations, CeOt growth ,82. Thesspecific .2 NPs. TiO2 NPs are extremely easy to agglomerate in aqueous solutions due to their large specific surface area, high surface energy and severe lack of coordination, making it necessary to ensure the stability of TiO2 NP dispersions in all relevant plant studies. Interestingly, it was found that the presence of tetracycline (TC) could significantly reduce the accumulation of Ti4+ released by TiO2 NPs in branches and roots [Arabidopsis could also mitigate chloroplast oxidative damage caused by low doses of TiO2 NPs through autophagy [Although it is generally accepted that oxidative stress (ROS) could be one of the direct sources of nanomaterials’ toxicity, relevant experiments showed that changes in ROS may not be the direct cause of the toxic effects of TiOnd roots . Furtherutophagy . These fArabidopsis in several other ways. For example, CuO NPs, CeO2 NPs and ZnO NPs can disrupt the energy transfer or oxidation from the photosystem to the Calvin cycle and reduce the gas exchange dynamics [2+ instead of Mg2+ in the chlorophyll center, and exhibit tissue specificity and concentration dependence. These observations corroborate experiments from phytohormone analysis [Arabidopsis leaves through the particles themselves to further disrupt the cystoid membrane structure, reduce chlorophyll content and inhibit plant growth [Additionally, MNPs have been found to affect the photosynthetic efficiency of dynamics . Furtheranalysis ,84. Ag Nt growth .Arabidopsis are complex. Sperdouli et al. exposed Arabidopsis leaves to CuZn NPs via foliar spraying and found that the photosystem II (PSII) function of young leaves was negatively affected, which could be attributed to the MNPs impeding the photosynthetic pathway by blocking the electron transport chain. In contrast, they observed a beneficial effect on PSII function in mature leaves and suggested that MNPs promoted the photosynthetic processes by improving light-harvesting complexes in plants [Arabidopsis using a gas exchange system, carbon isotope ratio and chlorophyll content analysis. They observed that enhanced stomatal conductance of Arabidopsis promoted photosynthesis and increased biomass by 38% after treatment with a 500 mg/kg concentration. In addition, the uptake of iron by the roots and leaves was increased -related genes and the alleviation of transcriptional gene silencing (TGS) in aerial leafy tissues were examined as genotoxic endpoints. It can be seen HR gene expression in aerial leaf tissue was upregulated, and TGS-silenced repetitive elements in aerial tissues could be observed. These observations suggest that the plant systemic response may involve distant induction of Ag NPs’ genotoxicity  are the two most common states. Previously, it was hypothesized that Ag+ release was responsible for Ag0 toxicity [+ and Ag NPs on Arabidopsis, the authors obtained inconclusive results. Studies on plant metabolism have confirmed that while both Ag NPs and Ag+ could induce glycolysis and affect the TCA cycle and aspartate family pathways, there are some metabolic changes  that occur only upon Ag NPs treatment. By comparing the differences in Ag NPs and Ag+ stresses, 111 genes responsible for the response to fungal infection, anion transport and biological functions associated with the cell wall/plasma membrane were found to be unique to Ag NPs at the Arabidopsis genome level  and monovalent silver  are t2O3 NPs stimulated the transcription of Arabidopsis root-development-associated genes and nutrient-related genes to promote root growth, but observed conflicting results when the same concentrations of Al ions were used because Al ions were highly toxic to plant growth and photosynthesis, and caused severe oxidative stress [Due to their different solubility, MNPs have been shown to affect plant growth and development and gene expression by releasing metal ions or attaching around plant tissues. Jin et al. found that high concentrations of Ale stress .Arabidopsis under hydroponic conditions to promote the degradation of microtubulin monomers, which in turn affects the plant’s cell division [Arabidopsis’s genomic instability by cooperating with other environmental stresses [Arabidopsis. Changes in sugar content, chlorophyll content, DAB and NBT staining and the antioxidant defense system showed that ZnO NPs were toxic to all genotypes of Arabidopsis [2+ is a hot topic of current research. Wan et al. compared the transcriptomic response of Arabidopsis roots to ZnO NPs, bulk ZnO and ionic Zn2+. They observed that the similarity of the transcriptional profiles and the increased number of transcripts with increasing concentration of Zn2+ in the culture medium suggested that the release of Zn2+ could be contributing to the toxic effects of ZnO NPs on the plant at the molecular level Arabidopsis used experimental settings that differed considerably from actual environment settings. For instance, the medium used was water or sandy soil rather than actual soil. The treatment time was comparatively shorter than that observed in nature, and Arabidopsis was exposed in a relatively single manner to MNPs, i.e., the roots were mainly exposed to the soil in the presence of MNPs, and thus, the effects of using leaf sprays or hydroponics remain to be determined.Currently available literature that investigated the effects of MNPs on (ii)The physicochemical properties, treatments and growth stages of the MNPs used in the experiments differed among studies. Therefore, the source of nanomaterials, preparation methods, testing equipment selection and design of exposure conditions should be standardized.(iii)The internalization of metal ions produces different levels of toxicity to plants than direct ingestion of metal ions. The toxic effect seems to be concentration-dependent. Further, as the toxic effects from MNPs could be from the released metal ions alone or the combined effect of nanoparticles themselves, the source of toxicity should be identified.(iv)Arabidopsis should be designed in a way that combines the traditional toxicological research methods with histological techniques  to provide more accurate and in-depth elucidation of the mechanisms of MNP-based phytotoxicity.As studies at different levels of the plant’s response, i.e., subcellular, physiological and biochemical levels, are being performed, the interactions between MNPs and In summary, MNPs enter


Homocysteine remethylation to methionine is catalysed by methionine synthase activity which requires vitamin B12, regenerating methionine to allow synthesis of the universal methyl donor S-adenosylmethionine required for methylation and gene transcription regulation. The methyl-group donated for homocysteine remethylation comes from 5-methyltetrahydrofolate generated by the folate cycle, which allows tetrahydrofolate to be returned to the active folate pool for nucleotide biosynthesis. Therefore the integrated actions of the methionine and folate cycles, required to metabolise homocysteine, also perpetuate methylation and nucleotide synthesis, vitally important to support embryonic growth, proliferation and development. Dysregulated activities of these two interdependent metabolic cycles, arising from maternal suboptimal intake of nutrient co-factors such as folate and vitamin B12 or gene polymorphisms resulting in reduced enzymatic activity, leads to inefficient homocysteine metabolic conversion causing elevated concentrations, known as hyperhomocysteinemia. This condition is associated with multiple adverse pregnancy outcomes including neural tube defects (NTDs). Raised homocysteine is damaging to cellular function, binding to proteins thereby impairing their function, with perturbed homocysteine metabolism impacting negatively on embryonic development. This review discusses the “cross-talk” of maternal-fetal homocysteine interrelationships, describes the placental transport of homocysteine, homocysteine impacts on pregnancy outcomes, homocysteine and methylation effects linking to NTD risk and proposes a putative pathway for embryonic provision of folate and vitamin B12, homocysteine-modulating nutrients that ameliorate NTD risk.Homocysteine is a metabolite generated by methionine cycle metabolism, comprising the demethylated derivative of methionine. Homocysteine can be metabolised by the transsulphuration pathway to cystathionine, which requires vitamin B We present an overview of some key characteristics of homocysteine and its effects in pregnancy, describe maternal-fetal homocysteine relationships in human pregnancy, elaborate on homocysteine transport mechanisms across the placenta, outline the potential protein-modifying cellular effects of homocysteine, make reference to the nutrient-modulation of homocysteine with emphasis on folate and vitamin Bvia three potential metabolic pathways, as illustrated in 12  as a cofactor, 2) remethylation by betaine as methyl donor to generate methionine and dimethylglycine catalysed by betaine-homocysteine methyltransferase (BHMT) or 3) transsulphuration to cysteine catalysed by cystathionine β-synthase (CβS) requiring vitamin B6 (pyridoxine) as a cofactor. 5-MTHF for homocysteine remethylation is generated by the activity of methylenetetrahydrofolate reductase (MTHFR) which requires vitamin B2 (riboflavin) to produce flavin adenine dinucleotide as a cofactor for MTHFR enzyme activity. Hence, homocysteine is placed at the intersection of the transmethylation and transsulphuration conversion pathways that require involvement of B-group vitamins, with the joint sustained activities of the folate and methionine metabolic cycles vital to ensure efficient metabolic disposal of homocysteine during pregnancy. These metabolic interdependencies between the two cycles highlight the need for adequate maternal intake of methionine, methyl donors such as choline  activities  are likely to occur in parallel making it difficult to attribute specific effects to an individual homocysteine metabolite diseases .de novo gene methylation  We discuss a putative transport pathway for the embryonic provision of folate and vitamin B12 based on their properties as homocysteine-lowering micronutrients which confer embryonic developmental benefit that lowers NTD risk, although knowledge remains relatively scant.With this in mind, in the next sections we focus on two aspects related to NTDs: 1) Based on the synergistic interaction of folate and vitamin Bcapacity , we consde novo methylation has a key role in neural tube closure; ablation of the DNA methyltransferase 3b (Dnmt3b) gene gives rise to cranial NTDs in mice, yet this phenotype is not seen in Dnmt3a knockouts, which may relate to relatively high expression of Dnmt3b in the embryonic neural ectoderm and the elevating neural folds during neural tube formation  in DNA isolated from fetal brain tissue showed hypomethylation of the MGMT promoter was associated with an increased risk for cephalic malformations, especially among female embryos  sequences; methylation signatures at these sequences are sensitive to nutritional factors such as methyl precursor availability in early embryonic development . LINE-1 da cases . In caseda cases . This isda cases . DNA met embryos . However embryos . An inheIn this context, others have examined placental tissue, as another fetal tissue of interest, revealing differential patterns of placental gene methylation between normal (at 40 weeks) and spina bifida (27–41 weeks) affected pregnancies, with the caveat that these were not gestation-matched. DNA methylation patterns for genes were differentially altered in the placentas of affected pregnancies but, interestingly, differential DNA methylation was most prominently altered in the transcription start site regions of genes functional in neurological system development, cell development and differentiation . Yet, coHowever, whilst these collective observations implicate a role for methylation-dependent neural tube closure, this does not require the interdependent activities of the folate and methionine cycles and absent maternal or embryonic MTHFR activity does not impair neural tube closure, yet the functional activity of both cycles is crucial . Indeed,Whilst much emphasis has been placed on investigating DNA methylation, it should be remembered that altered methylation applies to other methylation acceptors such as proteins in the context of neural tube closure . For exaTaken together, elevated homocysteine as a marker of perturbed folate-dependent methionine metabolism, together with accompanying impaired methylation appears to constitute a dysfunctional locus that is implicated in the aetiology of NTDs. Although multiple downstream methylation targets may be impacted by altered methylation, the early co-ordinated processes involved in neural tube closure seems particularly sensitive to such imbalances in methylation, with methylation regulation of DNA, histones and other proteins all likely to play a key role, in concert with other modes of epigenetic regulation .12 is not clear, but eminently plausible if maternal plasma transudates contribute to its composition. The glands gradually regress over the first trimester, but, until at least 10 weeks, remain connected with the placental intervillous space and their secretions appear to contribute to the fluid contained therein until this pregnancy stage  transporters for these micronutrients to be expressed and distributed to the first trimester human placental villi as well as the human yolk sac; and 2) favourable concentrations of these micronutrients in maternal plasma-derived transudates or uterine gland secretions that enter the intervillous space for uptake by transporters localised to placental villi, and in the coelomic fluid for yolk sac uptake for embryonic delivery.In order for such a histiotrophic pathway scheme to support the human embryonic provision of folate and vitamin BFOLR1 and SLC46A1 genes encoding FRα and PCFT respectively in human yolk sac  to its receptor (CD320), lysosomal export to the cytosol (LMBD1) and multidrug resistance protein 1 (MRP1)-mediated efflux , proton-coupled folate transporter (PCFT) and reduced folate carrier . By analyolk sac , which ayolk sac . A putatd efflux . Expressd efflux , again lyolk sac .12 in coelomic fluid relative to maternal plasma, which is not as apparent for folate, yet homocysteine concentrations in coelomic fluid are maintained at a low level, in accord with the notion that this exerts the protective advantage of maintaining the embryo in an environment that will limit the potential for homocysteine-induced oxidative stress (In support of the second concept, e stress  and tera12.Taken together, these findings are supportive of a postulate whereby the histiotrophic nutritional pathway, incorporating yolk sac-mediated uptake, plays a role in the embryonic provision of folate and vitamin B12 availability regulates homocysteine production. Perturbation of these metabolic cycles linked to one-carbon metabolism leads to the raised production of homocysteine and allied metabolites, which is associated with various adverse pregnancy outcomes including NTDs. The appropriate flux of one-carbon moieties through the folate and methionine metabolic cycles is essential for maintaining methylation capacity. The flux of one-carbon units through the methionine cycle, as well the partitioning of one-carbon donors from the mitochondrial glycine cleavage system to the folate cycle, is essential for neural tube closure necessitating the synergistic activities of both cycles (Optimal functioning of folate and methionine metabolic cycling, modulated by methionine, folate and vitamin Bh cycles . The flo12 have been shown to be effective in the amelioration of NTD risk, the loci of their action remains to be determined. A more complete understanding of the route by which the transport of such nutrients from the mother to developing embryo is achieved, would be insightful in supporting the mechanistic framework that underpins nutrient-based therapeutics in the amelioration of embryonic developmental anomalies.Given the intricate complexity and the multifactorial nature of human NTDs, delineation of methylated molecular targets and a more complete understanding of the regulatory mechanisms that maintain the metabolic interconnectivity that underpins methylation capacity, particularly in the embryonic period, remains an attractive area of research for developing future therapeutic approaches to reduce NTD risk. However, although a raised maternal provision of folate and vitamin B


Cholestatic diseases can be caused by the dysfunction of transporters involved in hepatobiliary circulation. Although pharmacological treatments constitute the current standard of care for these diseases, none are curative, with liver transplantation being the only long-term solution for severe cholestasis, albeit with many disadvantages. Liver-directed gene therapy has shown promising results in clinical trials for genetic diseases, and it could constitute a potential new therapeutic approach for cholestatic diseases. Many preclinical gene therapy studies have shown positive results in animal models of both acquired and genetic cholestasis. The delivery of genes that reduce apoptosis or fibrosis or improve bile flow has shown therapeutic effects in rodents in which cholestasis was induced by drugs or bile duct ligation. Most studies targeting inherited cholestasis, such as progressive familial intrahepatic cholestasis (PFIC), have focused on supplementing a correct version of a mutated gene to the liver using viral or non-viral vectors in order to achieve expression of the therapeutic protein. These strategies have generated promising results in treating PFIC3 in mouse models of the disease. However, important challenges remain in translating this therapy to the clinic, as well as in developing gene therapy strategies for other types of acquired and genetic cholestasis. Cholestatic diseases are based on bile dysfunction due to defects affecting bile synthesis or secretion. These processes involve a wide range of enzymes and membrane transporters involved in hepatobiliary circulation. According to its origin, cholestasis can be classified into two main groups: acquired cholestasis and genetic cholestasis .Most cholestatic diseases are acquired, presenting a dysregulation of the hepatobiliary transporters as a consequence of an adaptive and protective response to bile acid (BA) accumulation in the liver. This regulation is multifactorial, involving different elements such as hormones, BAs, proinflammatory cytokines, and drugs. These different factors mediate the activation of transcription factors that regulate the expression of export pumps, which promote the reduction of intracellular BAs by their excretion in the urine, resulting in the detoxification of the liver . AcquirePBC and PSC are classified as autoimmune diseases of the hepatobiliary system, characterized by the presence of antimitochondrial antibodies, portal inflammation, and an immune-mediated destruction of intra- and extra-hepatic bile ducts ,5. CliniICP, which is the most common disorder of the hepatobiliary system, is characterized by high serum BA levels in the third trimester of pregnancy that cause severe pruritus. In the development of this cholestatic disorder, high levels of gestational hormones, such as estrogen and progesterone, play a major causative role, while genetic factors may also be involved. Although symptoms disappear after childbirth, the biliary disorder can often recur during future pregnancies .Biliary atresia is a rare liver disease affecting the bile ducts, resulting in the main cause of neonatal cholestasis. The etiology of this biliary disorder is unknown. In some cases, the origin is thought to be due to an exacerbated autoimmune response in the bile duct epithelium as a consequence of a viral infection or due to toxin-induced injury after birth . In otheFinally, drug- and inflammation-induced cholestasis are closely related. Both drugs and proinflammatory agents can induce cholestasis following inhibition of hepatobiliary transporters but rarely result in severe liver injury. These types of cholestasis have an immunological origin mediated by proinflammatory cytokines directed against the bile duct epithelium that can alter BA secretion . ATP8B1, ABCB11, ABCB4, tight junction protein 2 (TJP2), NR1H4, and Myosin VB (MYO5B) genes are known to be the cause of PFIC 1-6 types, respectively (FIC1) gene cause the loss of the asymmetric distribution of phospholipid content in the canalicular membrane, leading to membrane destabilization and reduced BA transport, resulting in their accumulation in hepatocytes, causing cholestasis. Mutations in the ABCB11 gene can result in PFIC2 due to the absence of a functional bile salt export pump (BSEP) protein, which also leads to toxic accumulation of BA in hepatocytes. In PFIC3, mutations in ABCB4 cause multidrug resistance protein 3  deficiency, which results in low levels of phosphatidylcholine (PC) in the bile, which is needed to form micelles and neutralize the toxicity of hydrophobic BAs, resulting in damage to the epithelium of bile canaliculi. Mutations in TJP2 lead to the misdistribution of claudin tight junction in canaliculi, resulting in bile leakage and subsequently in PFIC4. PFIC5 is due to mutations in the NR1H4 gene that cause deficiency in farnesoid X receptor (FXR), resulting in a reduction of BSEP and ABCB4 expression and the accumulation of toxic BAs in the hepatocytes. Finally, mutations in MYO5B interfere with the processing of normal intracellular trafficking of BSEP, reducing its expression and activity at the canalicular membrane, which results in the accumulation of toxic BAs in hepatocytes, giving rise to PFIC6 .,52.51,52Recently, there has been great interest in developing drugs that are able to interrupt the enterohepatic circulation in a non-invasive manner for cholestatic disorders. The four transporters that allow circulation of BAs between the liver and intestine are the apical bile salt transporter , BSEP, the sodium-taurocholate cotransporter polypeptide (NTCP) and the basolateral organic solute transporter (OST) . The inhFurther additional pharmacotherapeutic approaches for the treatment of cholestatic disorders are being explored. Peroxisome proliferator-activated receptor (PPAR) agonists and fibroblast growth factor (FGF) analogues have been shown to be effective for diseases such as PBC and PSC . ActivatAlthough the pharmacological strategies mentioned above significantly improved the pathology of cholestatic diseases and the quality of life of the patients , they doGene therapy involves the addition, removal, or modification of the genetic material of an individual in order to treat a disease . Its effGene therapy has emerged as a promising approach to achieve safe, stable, and efficient long-term correction for a wide range of genetic diseases , includiSince no definitive treatment has yet been developed for some acquired hepatic cholestasis, such as PBC and PSC, there is a great need to identify novel therapeutic alternatives that can reduce fibrogenesis and potentially prevent the development of chronic liver injury, making genetic-based treatments an attractive strategy to achieve sustained long-term therapeutic effects.4), have been utilized [To generate animal models of acquired cholestatic disorders, interventions including bile duct ligation (BDL) and the induction of cholestasis by drugs, such as estrogens and carbon tetrachloride -expressing plasmid has demonstrated attenuation of hepatocellular apoptosis and liver injury in rats with BDL. IGF-1 promotes amelioration of cholestatic disease through activation of the phosphatidylinositol-3-kinase pathway, the inhibition of glycogen synthase kinase-3 beta, and the blockade of caspase-9 cleavage. Additionally, inactivation of hepatic stellate cells has been observed, which may explain the notable improvement in the degree of liver fibrosis . Reducing oxidative stress has been shown to be a therapeutic target for acquired liver cholestasis. For example, Adv-mediated mitochondrial superoxide dismutase (SOD) gene delivery leads to a reduction in liver injury by avoiding the formation of oxygen free radicals derived from the accumulation of hydrophobic BAs and preventing the release of proinflammatory cytokines, such as TNFα and TGF-β, in mice with BDL . SimilarAnti-fibrotic therapies for cholestatic disorders via reducing pro-inflammatory factors tend to promote collagen degradation and thus reduce the degree of liver fibrosis. Adv vectors expressing the urokinase-plasminogen activator (uPA) gene resulted in a slight reduction of liver fibrosis, leading to a partial improvement of liver histology in rats with BDL associated with the activation of metalloproteinases that trigger collagen degradation ,98. AddiFinally, Adv-mediated hepatic delivery of aquaporin-1 (AQP1) has shown an improvement in the bile flow of estrogen-induced cholestatic rats . In factThus, gene therapy approaches may offer a new avenue for the development of novel treatments for acquired cholestatic disorders.Gene therapy for the treatment of inherited hepatic diseases has garnered a great deal of attention after demonstrating that AAV vectors expressing human coagulation factors IX and VIII in the livers of patients with hemophilia B and A, respectively, resulted in a sustained therapeutic effect for more than three years . In fact-/-Ugt1a1 mice [Preclinical studies have shown promising results in animal models of Cerebrotendinous xanthomatosis (CTX) and Crigler-Najjar syndrome type 1. In the first case, the administration of an AAV8 vector expressing CYP27A was able to restore BA metabolism and normalize the concentration of most BAs in plasma in a mouse model of CTX . Interes-/- mice . In this-/- mice . These pThe results observed in preclinical studies of Crigler–Najjar syndrome showed that one of the main limitations for gene therapy of genetic cholestatic diseases could be related to the loss of viral genomes associated with hepatocyte proliferation occurring in young patients . Gene therapy approaches for PFIC can be based on gene supplementation or gene editing strategies to modify and repair the affected genes. The implementation of gene therapy for the different types of PFIC has some limitations. Firstly, in some types of PFIC in order to achieve stable and long-term therapeutic efficacy, it could be necessary to transduce most of the hepatocytes, which may require the use of high doses of the viral vector with the concomitant safety concerns ,108. Sec-/-Abcb4 mouse models with a range of phenotypes depending on the mouse strain [Although the loss of viral genomes could be a problem for most inherited cholestasis, ABCB4 deficiency, which causes PFIC3, has certain advantages over other PFIC types for liver gene therapy. For example, previous results using hepatocyte transplantation in a mouse model of PFIC3 showed that engraftment of 12% of healthy hepatocytes was enough to achieve therapeutic efficacy . This eve strain .-/-Abcb4 mice that were challenged with a BA-enriched diet to increase liver toxicity due to their mild phenotype. Treatment with an AAV8 vector expressing ABCB4 demonstrated long-term efficacy by preventing the increase of serum transaminases and the loss of biliary PC levels after BA challenge [-/-Abcb4 mice, which have a clinically relevant phenotype characterized by high serum levels of bile salts and transaminases, hepatosplenomegaly, and liver fibrosis [ABCB4 sequence downstream of the liver specific alpha-1 antitrypsin (AAT) promoter resulted in stable and long-term correction of PFIC3 by improving all disease markers. Interestingly, this therapy was not only able to prevent disease progression in young mice (two-week-old), in which symptoms had not yet developed, but also in older mice with an established phenotype (five-week-old and sixteen-week-old mice). The therapeutic effect was dose dependent, and it was observed that restoration of biliary PC levels above 12–13% (over 4000 µM) of wild-type levels was enough to have a curative effect. This indicates that PFIC3 could be treated even if only a small fraction of hepatocytes were transduced, in this way resembling gene therapy of other diseases like hemophilia B, in which therapeutic effects can be obtained with a small percentage of transduced hepatocytes. In our study, the therapeutic threshold was achieved with as little as 2–3% of wild-type ABCB4 expression levels [The first study tested gene therapy in C57BL/6 hallenge . In a sefibrosis . In thisn levels . Interesn levels . -/-Abcb4 mice [-/-Abcb4 show similar levels of serum biomarkers as the FVB -/-Abcb4 mice, but with a faster progression of liver fibrosis, leading to early development of primary liver cancers as well as an earlier onset of other complications, such as portal hypertension [ABCB4 mRNA-LNP injections were able to restore ABCB4 expression and biliary PC levels (~42% of wild-type levels), as well as improve serum biomarker levels, liver fibrosis, and hepatomegaly [Recently, a preclinical study based on LNP-encapsulated mRNA therapy was able to transiently reverse the disease phenotype in BALB/c -/- mice . BALB/c rtension . Five retomegaly ,115. How-/-Abcb4 mice. A single dose of the hybrid vector in neonates demonstrated the recovery of biliary PC levels and normalization of serum biomarkers. Additionally, the hybrid AAV-piggyBac treatment prevented biliary cirrhosis and reduced tumorigenesis [Using this type of approach, Siew et al. tested PFIC3 correction by the use of an integrative hybrid vector based on the expression of a piggyBac transposase and an AAV8 vector containing a piggyBac ABCB4 expression cassette in FVB igenesis . However-/-Abcb4 mice [HNF4A mRNA encapsulated with a biodegradable lipid restored the metabolic activity of hepatocytes in FVB -/-Abcb4 mice, leading to a robust inhibition of fibrogenesis [Although gene supplementation or correction of the affected gene is the most straightforward gene therapy strategy for PFIC3, several studies have shown that it is also possible to treat this disease by altering the expression of other genes that are involved in this pathology. One example is the delivery of vectors that express genes that contribute to the attenuation of liver fibrosis, such as ACE2 and HNF4A, as described in -/- mice . Moreoveogenesis . -/-Abcb4 mice. LIX1L regulates the levels of miR-191-3p, a microRNA that downregulates transcription factor liver receptor homolog-1 (LRH-1), thereby inhibiting Cyp7a1 and Cyp8b1 expression, two enzymes required for BA synthesis. Based on these data, Li et al. [-/-Abcb4 mice by direct repression of LRH-1 expression, thereby reducing de novo BA synthesis [-/-Abcb4 mice, knock-out of NK1R has been shown to decrease the levels of miR-31 and of proinflammatory molecules such as TFG-β1, resulting in the reduction of liver inflammation and fibrosis [A novel approach that could be used to treat cholestatic diseases is based on the regulation of BA synthesis and homeostasis. It has recently been described that Limb expression 1-like protein (LIX1L) is increased in the liver of patients with cholestatic diseases and that the normalization of its expression alleviates cholestatic liver injury in different cholestatic mouse models, including FVB i et al. , recentlynthesis . Anotherfibrosis . These tTJP2-deficient animal models available to test the feasibility of gene therapy for PFIC4 [MYO5B gene that affect the motor domain but do not result in complete deficiency of the protein [-/-Abcb11 mice in a C57BL/6 background represent the closest model to the patient disease phenotype, showing a drastic decrease in bile salt content in the bile that leads to increased levels of serum transaminases, liver fibrosis, and hepatomegaly, with these changes being more severe in females than in males [For other types of PFIC, although gene supplementation using vectors expressing the specific mutated gene is also an option, there are certain barriers that make the development of these treatments more challenging than for PFIC3. For example, patients with PFIC1, PFIC4, PFIC5, and PFIC6 have extrahepatic manifestations that cannot be rescued by liver-targeted gene therapy ,120. In or PFIC4 . Likewis protein . In the in males . HoweverFinally, the loss of transgene expression by hepatocyte cell division is a drawback for the use of non-integrative vectors, such as AAV, in gene therapy of these inherited cholestatic disorders that need to be treated at very early ages, as only a few hepatocytes will maintain episomal AAV genomes . Unlike Due to the growing success of liver-targeted gene therapies and preclinical studies showing therapeutic efficacy against cholestatic diseases, such as PFIC3, the need to overcome challenges involved in taking these products from bench to bedside is even more critical.One of the main challenges that gene therapy of cholestatic disorders faces is the potential loss of therapeutic effect in pediatric patients. This could be due to a decrease of viral genomes as a result of hepatocyte divisions in a growing liver in the case of AAV-based therapies, or to the transient expression of non-viral vector-mediated mRNA delivery ,125. OthFirst, the administration of repeated doses of the vector could allow the maintenance of the therapeutic effect. However, this is only straightforward for non-viral vectors, such as mRNA-loaded nanoparticles, although it will greatly increase the cost of this therapy ,125. ForSecond, the combination of gene therapy vectors with pharmacological therapies, such as UDCA, could provide synergistic therapeutic effects, especially in PFIC3 patients with more severe pathology who do not respond to UDCA treatment . The useThird, the sequential therapy of non-viral vectors such as mRNA-loaded nanoparticles in pediatric patients with growing livers followed by the administration of a viral vector that allows safe and stable long-term expression of the transgene at an older age, or the combination of vectors that, after reducing liver injury, facilitate the long-term efficacy of gene therapy could be of interest.Fourth, the improvement of gene therapy vectors by codon optimization or incorporating promoters that allow a more potent expression of the transgene with the aim of reducing the viral dose required to achieve a therapeutic effect could function to reduce the risk of toxicity from high doses ,130. AltAnd finally, for those cholestatic disorders in which correction of the majority of hepatocytes for a long-term therapeutic effect is likely necessary, as in the case of some PFIC subtypes ,109, a pAlthough pharmacological therapies can be used to treat cholestatic diseases with milder phenotypes, they are less efficient in patients with a more severe pathology. As addressed in this review, alternative approaches, such as gene therapy, could represent a promising novel approach. To date, many preclinical studies using liver-directed gene therapy in clinically relevant animal models of both inherited and induced cholestasis have shown promising results. Although there are still many challenges for the implementation of these emerging treatments in the clinic, it is likely that some of these therapies will be approved in the near future, giving new hope for many cholestatic patients.


Inhibitory signaling in the brain organizes the neural circuits that orchestrate how living creatures interact with the world around them and how they build representations of objects and ideas. Without tight control at multiple points of cellular engagement, the brain’s inhibitory systems would run down and the ability to extract meaningful information from excitatory events would be lost leaving behind a system vulnerable to seizures and to cognitive decline. In this review, we will cover many of the salient features that have emerged regarding the dynamic regulation of inhibitory signaling seen through the lens of cell biology with an emphasis on the major building blocks, the ligand-gated ion channel receptors that are the first transduction point when the neurotransmitter GABA is released into the synapse. Epilepsy association will be used to indicate importance of key proteins and their pathways to brain function and to introduce novel areas for therapeutic intervention. Epilepsy is a chronic neurological disorder characterized by repeated, unprovoked seizures. Seizure prevalence in those with epilepsy can range from one seizure every few months, to hundreds of seizures a day, the latter of which is usually seen in cases of genetic epilepsies . Yet, evNeurological disorders characterized as epilepsy can emerge throughout the lifespan, with cases of genetic epilepsy syndromes emerging in the first few years of life, childhood-restricted epilepsies, and new onset epilepsy in adulthood . FrequenThough numerous classes of medications have come into use for epilepsy in the past several decades, nearly all of them come with a heavy load of side effects that may exacerbate symptoms co-morbid with epilepsy such as mood disorders and learning and memory difficulties . AdditioARs)  . Furtherepilepsy .Replication of these studies, along with more recent immunohistochemical datasets from surgically resected tissue of patients with intractable epilepsy, have produced inconsistent findings that often do not validate earlier reports . AdditioARs and their efficacy as the brain’s major inhibitory sensor. We will explore regulation of (1) GABA receptor trafficking, stabilization, and localization, (2) GABAAR subunit composition (with associated changes in channel properties), (3) inhibitory synapse formation and elimination, and identification of (4) vulnerable GABAergic neuronal populations. Additionally, we will provide examples where a particular nexus of control is known, or proposed, to be dysregulated with epilepsy, either in animal models or in tissue samples collected from human epilepsy patients. Finally, we will discuss how the proliferation of single cell technologies and genetic disease models may allow for a more refined understanding of inhibitory brain dynamics that impact seizure disorders and how this may inform the development of more targeted therapies.Over the past 20 years there has been an expanded exploration into the basic mechanisms of inhibitory plasticity. A complex and dynamic signaling landscape has emerged that contains unique cell populations which deliver GABA into the synapse and a tightly controlled feedback system that regulates its receptors in discrete cellular compartments of specialized neuronal populations. In this review, we will examine various mechanisms that have been shown to alter inhibitory signaling, with a special focus on GABAARs to exert an inhibitory effect in neurotransmission, they must be located within the plasma membrane, either at the synapse (synaptic) or away from the synapse (extrasynaptic). Given the need for fine-tuned control of inhibitory signaling, neurons possess numerous mechanisms that they use to dynamically regulate GABAARs at the cell surface , and the phosphorylation status of a particular subunit, determining whether they are recycled back to the cell surface membrane, or targeted to the proteasome for degradation , and Golgi glycoprotein 1 (Glg1). However, only the presence of CLPTM1 altered GABAergic currents, leading to decreased amplitude of GABAAR mediated currents, but having no effect on the frequency of currents. Importantly, by preventing the progression of GABAARs out of the ER, and to the plasma membrane, CLPTM1 modulates both tonic and phasic inhibition.Trafficking of GABAmembrane . The γ2 ilepsies . In facteticulum . Interessynapses . A tandeARs to the plasma membrane comes from proteins that play a role in promoting re-insertion of receptors that have undergone endocytosis . This important study also highlighted the specificity of the HAP1- GABAAR subunit interaction which occurs with β1, β2, or β3 subunits. Given that all GABAARs will have one of these β subunit subtypes, HAP1 could have an important role in the receptor recycling of most GABAARs in the nervous system  , leading to de-phosphorylation of key residues on the subunit. Due to this dephosphorylation event, the β3 is no longer protected from association with endocytic machinery, mainly the adaptor protein AP2 . Ultimat(IPSC’s) .AR endocytosis plays a critical role is in activity dependent trafficking, both under physiologic and pathologic conditions  , and even inhibitory synaptogenesis (One important setting in which GABAnditions . Perhaps(NMDARs) . Part ofogenesis .ARs at the cell surface. This receptor-dependent potentiation of inhibitory synaptic plasticity relied on NMDAR-mediated calcium influx, and the activation of Calcium/Calmodulin-dependent protein kinase II (CAMKII). The investigators also identified a necessary role for N-Ethylmaleimide sensitive factor (NSF), GABA Type A receptor-associated protein (GABARAP), and Glutamate receptor interacting protein (GRIP) in the insertion of new synaptic GABAARs in response to NMDAR activation , Chiu and colleagues identified a form of inhibitory potentiation at dendrites that occurs in response to calcium influx through NMDARs , was required, rather than simply CAMKIIa activation from any calcium source. A role for CAMKII in iLTP was also demonstrated in a 2014 report that focused on mechanisms leading to increased clustering and the stabilization of GABAARs at synapses, again in the setting of iLTP. The investigators found that increased clustering of GABAARs at synapses relies on CAMKII-mediated phosphorylation of Ser383 in the β3 subunit of GABAARs, leading to recruitment of gephyrin from extrasynaptic sites to synaptic. Here, gephyrin mediates not only the increased recruitment of GABAARs but their stabilization at synapses, playing an essential role in the long-term potentiation of inhibition   .in vivo studies in the lithium chloride-pilocarpine SE model report that phosphatase inhibitors prevent the activity-dependent trafficking of γ2 subunit-containing GABAARs away from the synapse, suggesting that indeed, calcineurin-dependent dephosphorylation of γ2 promotes dispersal of γ2-containing GABAARs with increased neuronal activity , glycogen synthase kinase 3 Beta (GSK3B), and cyclin-dependent kinase 5 (CDK5). Many of the exogenous factors that disrupt gephyrin clustering likely act through these pathways, including estradiol/estrogen and brain-derived neurotrophic factor (BDNF) . Given tsynapses . In a reassembly .ARs at the synapse, as gephyrin-independent clustering of γ2-containing GABAARs has been identified. In a search for potential auxiliary subunits associated with GABAARs, like those previously seen and defined at excitatory synapses, lipoma HMGIC fusion partner-like 3 and 4 [referred to as the GABAAR regulatory LFRPL (GARLH family proteins that contain LH3- and LH4-like protein)] were identified as putative auxiliary GABAAR subunits that act as alternative scaffolding molecules at GABAergic synapses , serves to stabilize GABAARs at synapses by interacting with the γ2 subunit, in association with the GARLH protein LH4-like (see above) that helps to stabilize γ2-containing GABAARs at the synapse  leads to a decrease in gephyrin clustering , a protein involved in stabilization of extrasynaptic GABAantile 9 . PCDH19 currents .ARs in brain inhibition is influenced not only by their presence at the plasma membrane, as opposed to the abundant intracellular pool of GABAAR subunits, but critically whether they are located synaptically or extrasynaptically. Most importantly, there is a ready exchange between synaptic and extrasynaptic sites for receptors via lateral diffusion  could be found at the synapse . However, certain receptor subunit combinations were found more often at the synapse than others and intra-hippocampal kainic acid injection (IHKA) SE mouse models.In addition to subunit composition, specific interacting partners of GABApartment . Also imraplegia . In theiARs, it resulted in greater localization of these receptors to the postsynaptic membrane. The functional response to altering protrudin levels likely stems from its role in exocytosis, potentially through its physical interactions with the GABAAR and GABARAP . Using drugs that selectively block tonic inhibition, these same investigators demonstrated that protrudin overexpression increased the amplitude of sIPSC’s in both tonic and fast synaptic signaling. Additionally, they found that while protrudin did not impact overall levels of β2 or β3-containing GABA GABARAP . As discsynapses . In contmobility .ARs, some important progress has been made. In a study focusing on α5-subunit containing GABAARs that are normally located extrasynaptically, Torben and colleagues describe an anchoring protein, radixin, that typically serves to keep α5-containing GABAARs at the extrasynaptic membrane by preventing lateral diffusion. However, dephosphorylation of radixin, via Ras homologue family member A (RhoA)/Rho-associated protein kinase (ROCK)-mediated signaling disrupts the interaction between α5 and radixin, allowing α5-containing receptors to diffuse to the synapse as needed . The role of Shisa7 at inhibitory synapses was discovered incidentally when authors were trying to study the role of Shisa6–9 at excitatory synapses , and the ultimate properties and localization of their resulting ligand-gated channels. However, even after the synthesis of new GABAAR subunits, there are several additional layers of regulation at play that influence their ultimate functional contribution, and thus whether alterations in the transcript levels of unique GABAAR subunits will ultimately impact inhibitory transmission of the nervous system is still an active area of investigation.While the most dynamic regulation of inhibitory signaling in the brain may occur via a change in the trafficking, stabilization, and/or localization of GABAARs can play a critical role in the localization, and thus the resulting function of those receptors. Synaptic receptors are capable of mediating fast phasic synaptic transmission, whereas generation of additional receptors that localize to extrasynaptic spaces, especially α5γ and α4δ-containing GABAARs, will alter tonic inhibition. On a more refined level, changes to the subunit makeup of GABAARs can influence what endogenous and exogenous modulators exert effects on the receptors, and even impact the dynamics of the resulting IPSC’s produced with opening of the chloride channels. For detailed review of the major effects of subunit composition on GABAAR function please see the earlier reviews of Rabow et al. and Fisher and Macdonald  were some of the earliest transcriptomic datasets to be examined in epilepsy, both in resected patient tissue and animal models. Since the identification of these changes nearly 3 decades ago, significant work has illuminated the intracellular pathways that associate with epileptogenesis. To date, a range of molecular pathways have been implicated in epilepsy, from the Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) pathways to tropomyosin receptor kinase B (TrkB) and its neurotrophin brain derived neurotrophic factor (BDNF), REV-Erb alpha (Rev-Erbα), mammalian target of rapamycin (mTOR), among others  and subiculum in the long-term after SE, once spontaneous seizures arise in these models  model of SE-induced TLE in mice, and rats, differ in which GABAe models . Additioe models . In somee models . Alterate models , 2015. Re models . HoweverARs, along with clear evidence demonstrating alterations in mRNA levels and protein expression of numerous receptor subunits in animal and human TLE cases, an important area of past and current research exists for the development of new epilepsy therapeutics based on identifying the intracellular pathways that contribute to the genome expression of GABRs. Work from our own lab, in collaboration with our colleagues in the Brooks-Kayal lab, has helped to identify the intracellular signaling that leads to the transcriptional alterations in α1 and α4 gene expression in the context of epilepsy  through sequences upstream of the InR can be upregulated via the binding of the c-AMP response element binding protein (CREB) in response to protein kinase C signaling that solidifies its role in learning and memory, as well as in synaptic plasticity  can convert the “On” response to “Off”. Interestingly, this can occur in response to c-AMP signaling or in the case of BDNF, through a specific activation of the Janus Kinase (JAK)/signal transducer and activator of transcription (STAT) pathway that is dependent on TrkB (GABRA4p and GABAR subunit α4 transcript and protein levels through induction of early growth response factor 3 (EGR3) via MAPK signaling  within the intracellular domain of the α2 subunit of GABAARs that promotes interactions between the receptors and their binding partners, collybistin and gephyrin, that helps cluster GABAARs to produce effective inhibitory synapses  significantly decreased GABAergic neurotransmission at DIV8, whereas shRNA-KD of ST3 at DIV2 did not, suggesting that NLGN2 is essential at that time but ST3 is not. Later in development, elimination of either ST3 or NLGN2 at DIV10 significantly decreased mIPSCs at DIV18, representative of more mature neurons, and the effects of KO were additive. MAM Domain Containing Glycosylphosphatidylinositol Anchor proteins (MDGAs), on the other hand, inhibit the role of NLGN2 in forming inhibitory synapses, as KD of these molecules in the adult brain led to increased inhibitory synapse formation and upregulation decreased the number of inhibitory synapses  serving as a key transcription factor that is responsive to local neuron activity levels and plays a role in determining the number of functional inhibitory synapses that are formed . AnotherWhen it comes to the elimination of inhibitory synapses, microglia are a key cell type involved in the process. During development, microglia play an essential role in synaptic pruning, disruptions of which have been implicated in various neuropsychiatric disorders, including autism and schizophrenia. In addition to this developmental function, microglia continue to play a role in refinement of synaptic connections into adulthood . In orde+ interneurons in the hippocampus are particularly vulnerable to insults , and calretinin (CR), serve an important role as a buffering system for transient dysregulation in intracellular calcium levels. Thus, in the context of excessive excitatory signaling, calcium binding proteins that can buffer more calcium may provide added resilience against such insults when compared to the neuronal subtypes that primarily contain calcium binding proteins with lower buffering capacity. Of the 3 primary calcium binding proteins that are known to define distinct sets of inhibitory neurons in the hippocampus, PV has the lowest buffering capacity, while CR has the highest, which may contribute to the almost invariant vulnerability of PVs of TLE . And yets of TLE . While Ctermined . In addie points . In conte insult . Taken t+ neurons that may be particularly susceptible to degeneration in TLE. As for transcriptomic alterations, subsets of many broad classes of inhibitory neurons, defined by the authors using known subtype-specific expression markers derived from literature, including PV+ neurons expressing SULF1, PV+ neurons expressing nitric oxide synthase 1 (NOS1), vasoactive intestinal peptide positive neurons expressing cerebellin 1 precursor, SST+ neurons expressing tachykinin precursor 1, and inhibitor of DNA binding 2 positive neurons expressing lysosomal associated membrane protein family member 5 and NOS1, displayed dramatic shifts between control and TLE cases, providing more detailed information than was previously possible regarding GABAergic neurons and their signaling in the setting of human chronic TLE.With the advent and large-scale application of single cell technologies, future research will be poised to identify selectively vulnerable classes of inhibitory neurons, whose protection may offer a new therapeutic avenue in epilepsy . AdditioIn parallel with technological advances in single cell RNA-sequencing, recent advances in spatial transcriptomics are allowing researchers to capture spatially resolved single cell or even subcellular level transcriptomic and proteomic data. These spatially resolved techniques are a particularly valuable resource in epilepsy, as they will allow for an in-depth investigation of whole circuits that are disrupted and detail how alterations in one brain region may relate to those in another on an epigenetic, transcriptomic, and proteomic level. Spatial approaches that give context to transcriptomic findings may indeed provide important insights into the progression of epilepsy from the hyperactivity of neuronal subsets to a circuit level disorder in which epigenetic or genomic changes may drive epileptogenesis, providing new strategies for therapeutic intervention.ARs, the primary receptors responsible for synaptic and extrasynaptic neurotransmission, as well as the formation, maintenance, and elimination of inhibitory synapses in both health and disease. In part, this progress has been informed by the genomic revolution, which in the past two decades has allowed for identification of specific gene mutations in an increased number of individuals with genetic epilepsies, the exploration of which have propelled our understanding of GABAergic signaling forward. In the same vein, the single cell and spatial transcriptomic revolutions of the past five years have already begun to illuminate advancing complexity in the identity and function of cell types throughout diverse organs, including the brain, and offer a rich opportunity for enhancing our understanding of GABAergic neurons in the context of neural circuits that are dysregulated in a plethora of brain disorders, including epilepsy. The promise of multiomics to bring together the regulation of the genome with the functional proteomic landscape that is carved into the brain’s connectome will open new opportunities to rethink the development of curative therapies to restore the capacity of the brain for rebalance using its dynamic and complex inhibitory signaling properties.Significant progress has been made in the past three decades to elucidate the various cellular mechanisms that allow for fine grained control over inhibitory signaling within the CNS. Great strides have been made in our understanding of factors regulating the formation, properties, stability, and localization of GABAAT generated the original text for the document which was edited in collaboration with SR. AT generated the figures for general information to synthesize key concepts. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The protein kinase Mps1 (monopolar spindle 1) is an important regulator of the Spindle Assembly Checkpoint (SAC), the evolutionary conserved checkpoint system of higher organisms that monitors the proper bipolar attachment of all chromosomes to the mitotic spindle during cell division. Defects in the catalytic activity and the transcription regulation of Mps1 are associated with genome instability, aneuploidy, and cancer. Moreover, multiple Mps1 missense and frameshift mutations have been reported in a wide range of types of cancer of different tissue origin. Due to these features, Mps1 arises as one promising drug target for cancer therapy. In this contribution, we developed a computational biology approach to study the dynamics of human Mps1 kinase interaction with isoflavones, a class of natural flavonoids, and compared their predicted mode of binding with that observed in the crystal structure of Mps1 in complex with reversine, a small-sized inhibitor of Mps1 and Aurora B kinases. We concluded that isoflavones define a chemical scaffold that can be used to develop new Mps1 inhibitors for the treatment of cancer associated with Mps1 amplification and aberrant chromosome segregation. In a broader context, the present report illustrates how modern chemoinformatics approaches can accelerate drug development in oncology. Cancer has become the second leading cause of death in the human population, contributing to one in six deaths worldwide. According to the World Health Organisation (WHO), the most common cancers are breast, lung, prostate, stomach, and colorectal . CurrentMps1 is a protein organised into two main domains, an N-terminal region harbouring a triple tandem arrangement of the tetratricopeptide repeat (TPR) motif and a C-terminal region that harbours the characteristic bi-lobal fold of a protein kinase ,15,16. IIn human Mps1, the activation loop is defined by residues Gln672 to Lys680 and this region is only partially visible in refined crystal structures due to its intrinsic flexibility . The adoMultiple small-sized inhibitors of Mps1 catalytic activity have been reported in recent years ,21,22,23Although currently no drugs to treat cancer that target Mps1 catalytic activity have been approved for their use in the clinic, some Mps1 kinase inhibitors have been advanced to clinical trials, such as the compounds termed BAY 1217389 and BAY 1161909 ,26. ThesUnfortunately, the traditional route for drug discovery and development remains expensive and time consuming, with an average timeframe of 10 to 15 years of intensive work required before the drug reaches the market . Yet, thThe clonogenic assay, also known as colony formation assay, is an in vitro cell survival assay that is based on the ability of a single cell to grow into a colony . This tyOne aspect of cancer research that deserves more attention is the significant differences in cancer incidence in different ethnic populations. In addition to underlying genetic factors, there is mounting evidence suggesting that diet can play an important role in oncogenesis. For example, Asian populations consume a significantly higher soy food diet compared to Western populations  and thisMoreover, it is encouraging that daidzein and glycitein, when administered alongside tamoxifen (an anti-oestrogen medication that blocks the activity of oestrogen and is prescribed to breast cancer survivors) may decrease the risk of cancer recurrence . These fMps1 inhibitors described in the literature are ATP competitive inhibitors. Most of them present adenine moiety and establish between 1–3 hydrogen bonds with amino acid residues that define the hinge region . In the first place, molecular modeling studies of the Mps1 kinase domain with some of the most effective inhibitors described in the literature  were carReversine is a small molecule with high affinity for the ATP site binding of Mps1. This interaction is stabilised through the establishment of three hydrogen bond interactions with the hinge loop residues (two H-bonds with Gly605 and one with Glu603) .However, reversine is a small molecule first reported as an inhibitor of Aurora B kinase that abolished the Aurora B-dependent phosphorylation of histone H3. Hence, the search for more selective inhibitors of Mps1 remains important. To this aim, we first carried out the exploration of a chemical library of compounds that are structurally related to reversine (CID 210332). For this, the PubChem database with a 0.9 Tanimoto similarity index was screened. Upon conformational analyses, all the compounds were subjected to the high-throughput virtual screening (HTVS) XP Glide docking protocol. Compounds with docking Glide scores better than −9.0 kcal/mol, predicted specific interactions with Gly605-Glu603 residues, and significant structural diversity were considered the most promising Mps1 kinase ligands .Additional criteria to further filter out candidates included the computational analysis of predicted ADME properties and the identification of potential structural promiscuous moieties or PAINS (pan-assay interference compounds) ,56 for eThe isoflavone genistein has been reported to interact with death-associated protein kinase 1 (DAPK1), and the structure of genistein in complex with death-associated protein kinase 1 (PDB ID 5auz) has been solved at high resolution  . AnotherThe mode of interaction of the Mps1 kinase domain bound to reversine (PDB ID 5ljj) was compared against the predicted mode of interaction with the isoflavonoids daidzein, genistein, glycitein, and fisetin .The predicted best energy of binding was for the following flavonoids: fisetin  and genistein . For comparison, the predicted best energy of binding for reversine was −10. 9 kcal/mol. Fisetin is predicted to establish 2 H-bonds with the amino acid residues Asn606 and Gly605 whereas the other three flavonoids are predicted to establish only one H-bond with residue Gly605. Because fisetin exhibited violation of the Lipinski rules, it was excluded from the selection process. Genistein was chosen as the best isoflavonoid as it presents a good Glide score as well as predicted physicochemical and pharmacological properties (see Genistein (CID 5280961) was used to identify new potential flavonoids as possible Mps1 kinase inhibitors from a chemical library of compounds  that are structurally related to this molecule. More than one thousand compounds were screened and analysed. Upon conformational analyses, all the compounds were subjected to the high-throughput virtual screening (HTVS) Glide docking protocol and then XP docking Glide scores better than −9.0 kcal/mol, and those that presented specific interactions with Gly605-Glu603 residues and showed chemical structure diversity were considered for selecting the most promising Mps1 kinase binders . The structural diversity of the best compounds obtained by molecular docking as potential Mps1 kinase inhibitors is presented in To further filter out candidates, ADMET  and PAINS (pan-assay interference compounds) were investigated for each flavonoid  under the ID 5LJJ  was usedReversine and the isoflavonoid genistein were employed to retrieve structurally similar compounds from PubChem . The criThe LigPrep module of the Maestro suite was used to select three-dimensional conformations of low-energy molecules from the aforementioned chemical database and the programme Epik was employed to predict pKa values in the 7.0–7.5 pH range. The OPLS4 force field implemented in Maestro was used for energy minimization of all compounds .Docking grids were generated using the receptor grid generation tool within Glide to ensure ligand screening was performed in the appropriate site of the Mps1 catalytic domain. This was followed by the use of the Glide module to carry out high-throughput virtual screening (HTVS) and extra precise (XP) docking.The HTVS and XP Glide-dock modules integrated within the Schrödinger package were used for HTVS and XP docking calculations ,73. The A set of physicochemical properties were investigated on selected compounds, including pan assay interference compounds (PAINS) ,56 calcuIn this contribution, we report the development of a computational biology approach to study the dynamics of Mps1 kinase interactions with small-sized molecules of the isoflavone class, which has led to the identification of new potential Mps1 kinase inhibitors with predicted favorable drug-like features. Further functional studies of the compounds described in this work using an experimental approach will be carried out against a panel of cancer cells of diverse tissue origin, which should pave the way to identifying lead chemotypes, and, ultimately, to developing more effective therapeutics for the treatment of cancer and possibly other malignancies associated with Mps1 overexpression, defects in chromosome segregation, cell division, and cell proliferation. In a broader sense, our study provides a perspective of how state-of-the-art computational biology approaches can be used in oncology to advance the design of more effective drugs for the treatment of cancer.