Homo sapiens has challenged our species’s ability to occupy mountainous landscapes that acted as physical barriers to the expansion into lower-latitude Sunda islands during sea-level lowstands. Tham Lod Rockshelter in highland Pang Mapha (northwestern Thailand), dated between 34,000 and 12,000 years ago, has yielded evidence of Hoabinhian lithic assemblages and natural resource use by hunter-gatherer societies. To understand the process of early settlements of highland areas, we measured stable carbon and oxygen isotope compositions of Tham Lod human and faunal tooth enamel. Our assessment of the stable carbon isotope results suggests long-term opportunistic behavior among hunter-gatherers in foraging on a variety of food items in a mosaic environment and/or inhabiting an open forest edge during the terminal Pleistocene. This study reinforces the higher-latitude and -altitude extension of a forest-grassland mosaic ecosystem or savanna corridor (farther north into northwestern Thailand), which facilitated the dispersal of hunter-gatherers across mountainous areas and possibly allowed for consistency in a human subsistence strategy and Hoabinhian technology in the highlands of mainland Southeast Asia over a 20,000-year span near the end of the Pleistocene.The late Pleistocene settlement of highland settings in mainland Southeast Asia by  During the process of dispersal of Homo sapiens out of Africa to ultimately reach Australia, mainland Southeast Asia (MSEA) is recognized as being a potential route of human migration prior to crossing through the Sunda Shelf during a period of glaciation . Such a corridor might have facilitated the rapid dispersal of early humans through the intermediate migratory region of SEA6. However, the range expansion of inland vegetation examining the northern limit of a savanna corridor during the LGM is not yet known in great detail and the past human–environment interactions in the highlands of MSEA remain barely understood. Paleoecological and paleoenvironmental studies of the late Pleistocene hominin-bearing faunas in MSEA are thus important to understand the early modern human mobility and migration patterns through the land corridor into island Southeast Asia (ISEA).Our species has spread worldwide and successfully occupied a diversity of extreme environments such as deserts, arctic environments, tropical rainforests, and high-altitude conditions during the late Pleistocene 126–12 ka) (26–12 ka 19). The earliest modern human fossils, dated to a minimum age of 63–43 ka, were recovered from Tam Pa Ling, up to 1170 m in elevation, in Laos17. The increasing number of archaeological records in Thailand and Laos also supports the idea that high-altitude karst settings such as caves and rockshelters  were often used by hunter-gatherers23. Chrono-cultural frameworks for human remains and stone artifacts recovered from many archaeological sites in MSEA have suggested a typical “Hoabinhian” techno-complex characterized by flexed burials, sumatraliths, large and small tools made on cobbles, with an age ranging from the late Pleistocene to the mid-Holocene (e.g.27). A highland area (a range of low mountains or elevated parts of the country) is topographically characteristic of northern Thailand, which shares borders with Myanmar and Laos. Tham Lod Rockshelter (TLR) in Pang Mapha District  situated on a mountainous karst landscape with the elevation of 640 m above mean sea level  and thermoluminescence (TL) dating methods on various material including charcoal, sediments, and freshwater shells collected along the stratigraphic section of TLR have provided measurements for the faunal age ranging from 34 to 12 ka29 , dated between 19 and 12 ka . Although carbon and nitrogen isotope measurements of dentine collagen of a few late Pleistocene mammals (38.4–13.5 ka) from the cave of Tam Hay Marklot in northern Laos have successfully been carried out36, stable isotope tracking on dentine or bone collagen of human and animal remains from several archaeological sites in MSEA has been rather limited likely due to their location under the tropical monsoon climate where warm temperature and humidity usually prevented the good preservation of biological molecules such as DNA and collagen . In the case of TLR, faunal remains exhibited poor collagen preservation as indicated by the low amounts of nitrogen content  of several dentine and bone samples  varies from 2.0 to 6.9% and falls mostly within the normal range of recent samples that do not show evidence of diagenetic effects   plants and for habitats of closed versus open canopies. In the humid tropics, the cut-off δ13C values of − 10‰ and − 2‰ are applied for the dietary category distinction between herbivorous ungulates 41. In terms of habitats, the cut-off δ13C values lower than − 13‰ refer to the browsers that occupied a closed forest canopy, while the cut-off δ13C values higher than − 10‰ indicate a mixed feeder and/or grazer in more open habitats . In terms of trophic relationships, all δ13C values of predators present in this study (i.e. Panthera tigris) are adjusted upward by + 1.3‰ for a direct comparison with those of herbivores43). In omnivores such as some primates (including humans), rodents (i.e. Hystrix), and ursids (i.e. Ursus thibetanus), stable carbon isotopes in tooth enamel are incapable of distinguishing between plant- and meat-based (or insect-based) diets and the carbon isotopic fractionation between their food and tooth enamel remains unclear. In this study, we keep reporting enamel δ13C values of omnivores without isotopic fractionation corrections, which may not allow us to compare directly the trophic relationships between omnivores and herbivores/carnivores. The isotopic enrichment of 13C between diet and bioapatite in omnivorous mammals varies among species likely due to differences in their body sizes and physiological, anatomical, and behavioural traits . However, the δ13C values of omnivores can reflect the dietary intake of the ultimate plant resources (C3 or C4)46.Carbon isotope results are interpreted based on estimated cut-off values for diets of Cδ18O carbonate of tooth enamel is commonly used to investigate the isotopic composition of ingested water, despite having been influenced by several important factors . As the oxygen isotopic fractionation of meteoric water is incorporated into the animal's tissues via obligate drinking and/or plant-derived intake, some principles are often applied to the study of fossil mammals within the same temporal and spatial coverage. Mammals frequently ingesting water through drinking are expected to have lower δ18O enamel values than those of drought-tolerant taxa48. Grassland-inhabiting grazers may have higher δ18O enamel values than forest-dwelling browsers49.The δ13C values of human and mammal tooth enamel displayed a median of − 4.3‰ and ranged from − 16.0‰ to + 4.7‰, indicating reliance on a broad range of ecosystems expanding from pure C3 to C4 vegetation and corresponding to dense forests to open environments   suggested the consumption of pure C3-based resources in dense forest environments. One peculiar sample with a C4 signal (− 1.9‰) suggests that some individuals were able to consume grasses or possibly ate chiefly grass-eating insects such as grasshoppers, as observed in the feeding habits of extant long-tailed macaques in Malaysia and Java51.Humans The δ13C values of human teeth  almost suggested the consumption of a combination between the higher amount of C3 foods (plants and/or C3 plant-dwelling preys) and some C4 items and/or the occupation in open forests . The most negative δ13C value of − 14‰ in the tooth sample collected from the layer 3 near the ground surface showed human reliance on pure C3-based resources in a closed forest canopy.Rodents The δ13C values of Hystrix sp.  suggest that porcupines consumed only pure C3-based resources in closed or open canopy forests, which were not as dense as where the macaques occupied (P < 0.05). The δ13C values of small indeterminate rodents  yielded a high variability in dietary intake and habitat use ranging from closed C3 to open C4 ecosystems (P > 0.05 between indeterminate rodents and humans).Omnivorous Carnivora Ursus thibetanus  might have had the primary diet of C3 food items  similar to that of extant populations52, and shared a closed habitat with the porcupines (Hystrix sp.) (P > 0.05) and some ruminants such as small-sized deer Muntiacus sp. and Axis porcinus. One sample of Arctonyx collaris (− 10.9‰) reflected its food preferences on C3 items such as tubers, roots, and small creatures52 and its occupation of an open forest landscape.Suids Samples of Sus scrofa  imply that their diet consisted of a mixture of C3 and C4 resources in an intermediate area between open and closed landscapes or sometimes had a pure C3 diet in an open forest. This also indicates that both types of vegetation were available at the same time and in a limited area.Predatory Carnivora The adjusted δ13C values of Panthera tigris  suggest that tigers were probably preying essentially on S. scrofa or might have been consumers of a wide range of prey species in both open and closed canopies.Elephants One sample of Elephas sp. exhibited a δ13C value of − 4.7‰, which suggested a mixture of C3 and C4 plants in an intermediate area between closed and open canopy landscapes.Rhinoceroses The rhinocerotid enamel samples  suggested a wide range of diets varying from pure C3 to C4 vegetation. Otherwise, it is possible that there were two separate rhinocerotid taxa in the locality, one C3 browsing species  and two C4 grazing individuals , as observed in some Pleistocene Southeast Asian sites53.Cervids The small-sized muntjac deer, Muntiacus sp. , had pure C3-plant diets and occupied a forest landscape, similar to the extant population of Muntiacus muntjak (Indian muntjac)52. The samples of Axis porcinus  indicated a pure C3 plant diet in more closed canopy habitats, compared to Muntiacus sp. The Eld's deer Rucervus eldii  had a pure C4 diet and occupied an open habitat. The sambar deer Rusa unicolor  indicated their primary utilization of mixed C3/C4 plants but sometimes pure C4 ones and had the occupation of open canopy habitats but more closed than where Rucervus eldii lived (P < 0.05). Unlike their modern representatives that have a habitat shift to more closed forests52, the late Pleistocene Rucervus eldii and Rusa unicolor in TLR were reliant on an open grassland landscape. It is apparent that substantial resource partitioning and minimized intergeneric competition occurred among TLR cervid communities, leading to the prevalent coexistence of four cervid genera/species during the late Pleistocene.Bovines Samples of wild water buffaloes, Bubalus arnee,  reflected a pure C4-plant diet. Two large bovid species, Bos javanicus  and Bos gaurus , consumed substantial amounts of C4 plants, similar to Bubalus arnee (P > 0.05). The samples of undetermined species, Bos sp.  also focused on pure C4 vegetation in an open habitat.Caprines Stable isotope data of two goral taxa  and one serow species (sumatran serow Capricornis sumatraensis) from TLR were analyzed by54. Both Naemorhedus griseus  and Naemorhedus goral  showed the same dietary habits in foraging on mixed C3 and C4 vegetation and on pure C4 plants (P = 0.05)54. However, Capricornis sumatraensis  had a wider range of diets, compared to the two goral species (P < 0.05), reflecting its occupation of closed C3- to open C4-dominated landscapes54.δ18O values  or from the nonsimultaneous occurrence of fossils that represented a different age through the stratigraphic section. The pairwise Mann–whitney U test indicates that there are almost no statistically significant differences in median δ18O values between two TLR mammal taxa (P > 0.05), except for wild boars , compared to other mammal taxa within the same locality (P < 0.05). It is possible that these suids fed on more 18O- depleted foodstuffs  on the open forest/woodland ground surfaces58.Several samples from the same taxa/locality showed a high variability and relatively wide ranges of ues Fig.  that lik61, we analyzed five high-crowned molars of large bovids (Bos gaurus (n = 3), Bos javanicus (n = 1), and Bos sp. (n = 1)) from different stratigraphic layers of TLR to demonstrate seasonal patterns in diet and precipitation through the late Pleistocene  per yearene Fig. .Figure 4δ13C variation of 1.2‰ (from − 0.4‰ to + 0.8‰) for TLR-A3697, 0.9‰ (from + 0.5‰ to + 1.4‰) for TLR-A1677, 1.0‰ (from + 1.7‰ to + 2.7‰) for TLR-A1291, 3.0‰ (from − 2.1‰ to + 0.9‰) for TLR-A1207, and 0.8‰ (from + 1.1‰ to + 1.9‰) for TLR-A920, all of which indicate grazing habits on the same types of food items (pure C4 plants) throughout the course of almost 1 year during the terminal Pleistocene  for TLR-A3697, 2.4‰ (from − 6.5‰ to − 4.1‰) for TLR-A1677, 2.7‰ (from − 6.7‰ to − 4.0‰) for TLR-A1291, 2.1‰ (from − 8.9‰ to − 6.8‰) for TLR-A1207, and 2.4‰ (from − 4.4‰ to − 6.8‰) for TLR-A920. All the serially analyzed samples showed little seasonal variation in precipitation over several months to years through the terminal Pleistocene . As supported by similar isotope compositions of a contemporaneous mammal fauna from the slightly higher-latitude cave of Tam Hay Marklot in Laos36, we purport to document the possible extension of a latitudinal limit of the savanna corridor farther north than previously recorded during the LGM 28  indicated a dramatic shift in precipitation to the wetter climate condition starting around 16 ka (Heinrich event 1) and at the end of the Pleistocene (Younger Dryas)68  collected at the layer 3 of TLR (dated around 12 ka) showed alongside a shift in δ13C towards higher reliance on C3-based resources in a more closed habitat during the Pleistocene-Holocene transition . Despite the seemingly specialized occupation of coastal resources by other human populations in ISEA within Wallacea, stable isotope compositions of human and faunal tooth enamel in Alor suggested the possible availability of C4 resources used by late Pleistocene small mammals and hunter-gatherer populations (40–21 ka)90. All the evidence has suggested so far that more specialized hunting of arboreal and semi-arboreal mammals and the occupation of tropical rainforest habitats have commonly been favored among the late Pleistocene hunter-gatherer populations from these aforementioned regions. Unlike the long-established rainforest occupation by those late Pleistocene hunter-gatherers, our isotopic data provide direct evidence for human reliance on the different types of habitats (dated around 19–12 ka) in MSEA.Along the major routes of human migration from Africa to Australia and across SEA, recent studies have shown that tropical rainforests, previously thought as an unfavorable niche for the early human occupation31. Studies of lithic assemblages have shown that classical sumatralith forms such as flakes and radial cores made from metaquartzites or sandstones were found from TLR, while microliths indicative of specialized rainforest hunting by Homo sapiens, as discovered in the archaeological sites of Sri Lanka about ~ 45,000 years ago95, were absent in the area29. The analysis of ecological models applied to flaked stone artefacts in TLR have suggested that proximity to resources and climate change were important factors influencing Hoabinhian technology29. In addition, the stable isotope data obtained in the present study allow us to evaluate directly the degree of early modern human reliance on a broad spectrum of resources and to refine the relationship between the lithic assemblages and prevailing open environments in TLR. Environmental heterogeneity in highland Pang Mapha allowed Homo sapiens to have more opportunistic foraging on various types of preys and more available C3- and C4-based resources to exploit during the terminal Pleistocene. The population of late Pleistocene hunter-gatherers in Pang Mapha might have selected a settlement option at the edge of open forests where they could take advantage to exploit both closed and open canopy resources, not a particular area in one extreme direction of environmental ranges. Compared to the timing of microlithic records in South Asia, our study reinforces the late occurrence of the proliferation of microliths in highland MSEA. The high environmental heterogeneity resulted in low ecological stress in forcing and shaping behavioural adaptations among the late Pleistocene hunter-gatherers, allowing the persistence of similar subsistence patterns and stone artefact technology in the area for at least 20,000 years.Based on available zooarchaeological information on the taxonomic diversity, species abundance, and mortality profiles of successive faunal assemblages from TLR, it has been suggested that hunter-gatherers in the area adopted a generalized and mixed subsistence strategyHomo erectus and Homo florensiensis in ISEA , the degree and direction of ecological reliance between Homo sapiens and other hominin species on the broad spectrum of resources and environments might be questioned and require future studies. However, our findings stand in striking contrast to the specialized rainforest occupation among other contemporaneous hunter-gatherers in MSEA’s neighboring continents/regions. Our isotopic evidence not only suggests the asynchronous occurrence of specialized rainforest occupations among the hunter-gatherer populations in MSEA, but also documents diverse late Pleistocene human adaptations during the process of dispersal towards Wallacea and Sahul.As it is visible in the settlement of highland Northwest Thailand by early modern humans during the late Pleistocene, they have ventured into high-altitude mountains where the savanna ecosystem was present. Although the TLR hunter-gatherers had the mixed grassland and woodland adaptations comparable to other earlier hominins such as We analyzed a total of 139 bulk tooth enamel samples of humans and non-human mammals from the late Pleistocene archaeological sites of Tham Lod Rockshelter in highland Pang Mapha. Five high-crowned teeth of large bovids from TLR (45 samples) were selected for serial sampling. All teeth were collected along each well-dated layer from one stratigraphic side of the wall (west profile) in the Area 1 of TLR, in order to ensure the vertical placement of samples Fig. . SixteenFor preliminary measurements in the study, we performed a CNS elemental analysis to test the possibility of collagen preservation in bone and dentine samples from Tham Lod Rockshelter (Supplementary Table δ)–notation: X = [(Rsample/Rstandard) − 1]*1000, where X stands for 13C and 18O values and R is referred to 13C/12C or 18O/16O, respectively. The recorded delta values follow the international reference standards, “Vienna PeeDee Belemnite” (VPDB) for the carbon and oxygen. δ18O values relative to “Vienna Standard Mean Ocean Water” (VSMOW) are also given. The carbonate content (CaCO3%) was calculated using the ratio between amount of CO2 released by the reaction, as detected from the peak intensity for mass 44 and the weight of pure carbonate used as a standard, with an analytical error of 0.3%, based on the multiple analysis of reference enamel samples.Bulk enamel powder was sampled along the whole length of a tooth crown in order to obtain average isotopic signals over the time period of dental growth, while serial sampling was taken along a non-occlusal surface parallel to the growth axis and across its entire length (lengthwise from the top to the bottom of tooth crowns) on the labial side of lower molars. The isotopic pretreatment and measurements of samples were performed at the Department of Geosciences, University of Tübingen, Germany (see Supplementary Information 3 for more detailed information on the isotopic pretreatment and protocols). Stable carbon and oxygen isotopic values are expressed as the following standard delta  and between the species (Mann–Whitney U-test) (Supplementary Table 98.Using the Shapiro–Wilk test, our samples with unequal variances do not correspond to a normal distribution. In the case for which at least five samples are available, Supplementary Information 2.Supplementary Information 1.


This method has not been previously used for these viruses.Virus production is an essential part of virology. The quality of virus preparation, in terms of purification and yield, can affect the outcome of experiments. Purified virus is critical for assays, as contamination from cell culture supernatants may affect results. Purified virus is also needed for vaccine preparations. The traditional process of gradient purification of virus is time consuming, with multiple steps spanning over days, and produces significant amounts of biohazard waste. Developing a more efficient alternative that provides a purified virus product is highly desirable. In this study, we performed magnetic bead purification of Zika virus (ZIKV) and Mayaro virus (MAYV) with the Mag4C LV kit from OZ Biosciences. Currently, this kit is only indicated for use with lentiviruses and retroviruses. We found that we were able to successfully concentrate and purify both ZIKV and MAYV using the kit. Therefore, we concluded that the Mag4C LV kit provides a quick and simple alternative to traditional virus purification methods.  Specifications tableVirus production is an essential part of virology experiments and the quality of virus preparation (in terms of purification and yield) can affect the results of these experiments. Purified virus is critical for assays where contamination from the cell culture, e.g. cell culture medium additives, products of cell lysis , cytokines, and chemokines, may affect the results of the assays Magnetic beads can be used for many biological applications, such as immunoprecipitation •Minimal Eagle's Medium (MEM) •Newborn calf serum (NCS) •Penicillin/streptomycin •5-layer cell culture flasks •50 mL conical tubes •1.5 mL screw-cap tubes •15 mL conical tubes •1 mL pipette, 200 µL pipette and tips •Refrigerated centrifuge set to 4°C•0.22 micron vacuum filter/storage system •○Mag4C LV magnetic beads○Elution bufferMag4C LV kit •Magnetic separation rack •Biosafety cabinet•Incubator with 5% CO2 set to 37 °C•1X PBS(1)Seed Vero cells in 5-layer cell culture flasks and grow to confluence in complete medium (MEM + 10% NCS + 1% P/S).(2)Inoculate cells with MAYV or ZIKV and allow virus to multiply for 2 or 4 days, respectively.(3)Decant supernatant from multi-layer flasks into 50 mL conical tubes.(4)Centrifuge at 4 °C at 3500 rpm for 30 min.(5)Vacuum filter (0.22 µm) the supernatants and store at 4 °C.(6)Add 2 mL of clarified supernatant to a 15 mL conical tube.(7)Add 20 µL of Mag4C LV beads to the 2 mL of clarified supernatant.(8)Incubate for 30 min at room temperature.(9)Place tube on magnetic separation rack for 30 min.(10)At this point, the beads should be held on the wall of the tube. Remove the supernatant and save as a sample (leftover clarified supernatant) at −80 °C.(11)Remove the tube from the magnetic stand.(12)aAt this point, the sample can be transferred to a smaller tube if desired.Resuspend the beads in 200 µL of elution buffer.(13)Incubate for 10 min at room temperature.(14)Place tube on magnetic separation rack for 30 min.(15)Collect the supernatant (this contains the eluted virus) and save as a sample (eluted virus) at -80 °C.(16)Remove the tube from the magnetic stand.(17)Resuspend the beads in 100 µL 1X PBS and store as a sample (leftover beads) at −80 °C.Each sample collected during the magnetic bead purification process was subjected to protein estimation by bicinchoninic acid (BCA) protein assay, protein gel, western blot, and determination of the median tissue culture infectious dose (TCID50/mL) The qualitative detection of total protein quantities  was performed using protein gel analysis. 10 µg of protein per sample was separated on a 4–12% Bis-Tris gel  under reducing conditions. The protein gel results correspond to the BCA results; specifically, as more purification steps are performed, less excess protein is observed on the gel, while the virus-specific proteins remain. It is also demonstrated that there are negligible amounts of protein remaining on the beads at the end of the magnetic bead purification process, indicating that the majority of virus has successfully been eluted.The binding of virus to virus-specific monoclonal antibodies  was evaluated by Western blot. Western blot analysis of the ZIKV samples using the ZIKV specific antibody ZV54 showed that the antibody was able to bind to the purified sample, indicating the presence of proteins characteristic of ZIKV. The intensity of the band decreases as more purification steps are performed, indicating possible loss of virus, which is consistent with the titer data . Similar–1, indicating loss of virus particles during the purification process. The process was performed on small volumes for ZIKV  with minimum instrumentation need yet produces purified virus. However, the loss of virus with each purification step may limit the use of this approach where very high concentration and yield may be needed in downstream applications.Magnetic bead purification successfully separates ZIKV and MYAV from various impurities and contaminants , and theOpinions expressed herewith are those of the author(s) and are not necessarily representative of those of the USUHS, DoD, or the United States Army, Navy, or Air Force.


Our Monte Carlo-based inversion method identifies two scenarios to account for our data: an increase in erosion rate by 1–2 orders of magnitude between ~2.6 and 1.1 ka, or a discrete event stripping ~1–2 m of soil between ~1.9 and 1.1 ka. Coupled environmental and cultural factors in the Late Holocene signaled the onset of the pervasive human imprint in the Andean Altiplano seen today.Soil sustainability is reflected in a long-term balance between soil production and erosion for a given climate and geology. Here we evaluate soil sustainability in the Andean Altiplano where accelerated erosion has been linked to wetter climate from 4.5 ka and the rise of Neolithic agropastoralism in the millennium that followed. We measure in situ cosmogenic  14C, 10Be, and 26Al to reveal rapid soil erosion on the Andean Altiplano in response to Late Holocene climate change and the onset of agropastoralism.The assessment of soil sustainability in prehistoric times requires comparing millennium-scale erosion rates with geological background rates. Here, the authors apply in situ cosmogenic  Soil sustainability is a function of this long-term mass balance that culminates in a steady-state soil thickness for a given hillslope curvature as set by climate and geologic setting4. Changes to climate or land use can accelerate the loss of soil, diminish its biological productivity and compromise sustainability as erosion outpaces soil production. Past changes in surface erosion rate are a direct result of how soils respond to environmental change, but with systematic observations of soil erosion rarely more than a few decades long, the lack of data over timescales relevant to soil formation  has hindered a synoptic understanding of soil evolution where natural and anthropogenic forcing intersect.Hillslopes co-evolve with landscapes and vegetation communities to strike a balance between rates of soil production and soil erosion over geological timescales5. To this end, vital gains have been made by extracting sedimentary records from floodplains, lakes and estuaries7. However, this method relies on a close relationship between sediment source and sink, which can be compromised by sediment-transport filtering effects that bias the stratigraphic record8. A general difficuly for studies of soil sustainability lies in discriminating the earliest impacts of humans from climate forcing, albeit much evidence points to the coupling of these factors at least during the last few millennia11. Climatic variations since the last glacial–interglacial transition were a key driver of the technological and social transformations that spawned farming across multiple centres during the Neolithic14. This link implies humans were exploiting new capacities in their environment and it underscores connections between climate and the impacts of land use on soil, plants and the atmosphere18. Proponents of an ‘Early Anthropocene’ point to episodes of soil depletion during agriculture’s pre-industrial rise18. If the relationship between the earliest human-driven coupled with climate-driven impacts is a potential basis for assigning the Early Anthropocene, then assessment of soil sustainability across multiple centres of Neolithic agriculture points a way forward.Given that long-term soil production and erosion tend towards a balance, a compelling evaluation of soil sustainability is to quantify and compare erosion with geological background rates26. Some early studies directly link cultural development to climate and environmental change, suggesting massive landscape modification prior to the collapse of both the Tiwanaku (~1 ka) and Inca (~0.5 ka) civilizations29. Others have emphasized drastic soil erosion during the past century30. The debate is sustained in part, because no evaluation of soil sustainability has ever been conducted and the erosional dynamics arising from climate and land use remain unquantified.The Titicaca Basin on the Andean Altiplano is an early centre of agricultural development and its archaeology and palaeoclimate have long been in the research spotlight14C, 26Al and 10Be in hilltop soil and bedrock and in river sediments of the eastern central Altiplano, south of Lake Titicaca  allows resolving erosion rates over variable timescales employing the long-term 10Be-26Al chronometer (~104–105 yr) and the short-term 14C-10Be chronometer (~102–103 yr). If the two chronometers show different apparent erosion rates, we can deduce erosional transience over Holocene (anthropogenic) timescales34  is governed primarily by the nuclide half-life and production rate. We measured in situ-produced cosmogenic 37. A dry Early Holocene culminated with intense aridity at 7.9–4.5 ka38 followed by wetter conditions over the past few thousand years35 persisting until today. The wetter climate from about 4.5 ka caused a rising Lake Titicaca to overspill into the Desaguadero River at 3.6–3.5 ka39  transition spanned 3750 and 2940 cal. yr BP according to a Bayesian analysis of 14C ages from archaeological contexts44. The adoption of food production during this time of environmental change led to a significantly modified Altiplano landscape. Land clearing, tilling and irrigation for agriculture and pastoralism rapidly took hold, especially around Lake Titicaca, where consistent population growth prompted the development of increasingly large and intensive farming infrastructure including raised fields, irrigation channels, terraces and reservoirs45.Archaeological research has documented hunter–forager populations in the Andean Altiplano from the terminal Pleistocene and between 10 and 3.5 ka, although population densities remained low largely due to environmental variabilityDistichia tussock grasses and Polylepis and Buddleja shrub remnants21. Abundant traces of past and contemporary traditional agropastoral activity in the area46 are seen in extensive stone structures including livestock enclosures and low walls marking field boundaries , part of the Desaguadero catchment that straddles the eastern rim of the Altiplano ~150 km southeast of Lake Titicaca Fig. . Elevatiley Fig.  and is cn = 3) and soil (n = 6) to constrain point rates of surface lowering. The stony soils comprise rounded cobbles in a sandy matrix and, in places, platy fragments of siliceous hardpan overlying Cenozoic siltstone  are in good agreement . The 10Be-26Al apparent rates of long-term hilltop lowering vary according to bedrock erodibility with poorly lithified rocks of Cenozoic and Mesozoic age eroding faster  than the stronger Palaeozoic rocks . Palaeozoic clasts recycled into the younger conglomerates also fall among the slowest erosion rates . The 10Be and 26Al apparent erosion rates obtained from fluvial sediment samples span a range similar to the point rates (7.2 ± 0.6 to 20.2 ± 1.7 mm kyr−1).Apparent erosion rates obtained for the long-lived nuclides  are overall concordant with the 10Be-26Al rates  also agree with the 10Be-26Al rates. In contrast, the three other hilltop soil samples  plus all four fluvial samples reveal a pronounced offset between apparent erosion rates for 14C and those for the longer-lived 26Al and 10Be. The 14C apparent erosion rates of 140 ± 2 to 270 ± 23 mm kyr−1 (hilltop samples) and 202 ± 17 to 311 ± 26 mm kyr−1  are up to an order of magnitude faster than the corresponding 10Be erosion rates  a simple step change in erosion rate with an instantaneous shift at a specific time and (2) an abrupt erosional spike that rapidly removes soil of variable thickness at a specific time before returning to a constant pre-spike erosion rate. These limiting cases describe how a major landscape perturbation may have affected surface erosion rates. To test these two scenarios and delineate probable erosion histories, we apply a joint MCMC inversion model  show a ~40- to 150-fold increase in hilltop erosion rates between 2.6 and 1.1 ka  in both our hilltop soils and fluvial sediments suggests that a shared landscape-wide signal of transience has been transmitted from the hillslopes to the river system.Apart from erosional transience, other mechanisms have the potential to deplete 7 and references therein. When dealing with sedimentary archives, temporary sediment storage or even the dynamics of sediment transport itself can interrupt and distort the erosional and depositional record of events leading to a highly filtered stratigraphic record53.A key advantage of our multi-nuclide approach to quantifying soil erosion is that measurements are conducted directly at the sites of soil production. This makes it possible to evaluate accurately the balance between production and erosion without the need of an assumed coupling between sediment source and depositional sink—an assumption that is central to assessments of soil sustainability based on records retrieved from floodplains, lakes or estuaries10Be-derived erosion rate of 10.8 ± 7.6 mm kyr−1 (±1σ) averaged across 18 catchments including the study area   or 2110–1180 cal. yr BP (2σ) indicate that the silty floodplain deposits (up to 4 m thick) are direct counterparts to the accelerated erosion of the hillslope soils  Fig. . Soils s54. From an analysis of palaeohydrological proxies along a north–south transect, Abbott et al.55 suggest that wetter conditions came to the southern Titicaca basin ~2 kyr later (~2.5 ka) via a progressive southward shift in wet-season convection. This points to a convergence in the timing of climatic amelioration and flooding along the Desaguadero valley over the period ~3.5 to 2.5 ka. In response, the sparse plant cover that developed under aridity would have become denser, thereby reducing erosion in the uplands58. The onset of soil thinning during the interval ~2.6 to 1.9 ka followed this chain of events, although the temporal uncertainty allows for a lag of up to several centuries after the upswing in effective moisture  a wetter climate from the mid-Holocene with the overflow of Lake Titicaca and watering of the Desaguadero River valley and (2) the transition from hunter–forager subsistence to agropastoralism in the Formative (Neolithic) Period at ~3.5 ka  already existed at the time of the demographic expansion down the Desaguadero valley. The best estimate of the agropastoral transition, 3.75–2.94 ka44, implies that soil-thinning was preceded by several centuries to a millennium or so. This delay (if not a function of imprecise dating) possibly reflected the demographic response time to a boost in resources62, or perhaps an initial phase of sustainable farming prior to the onset of practices that led to irreversible soil loss on the uplands.Archaeological research in the central Altiplano demonstrates the adoption and development of camelid pastoralism supplemented by quinoa and tuber cultivation from ~3.4 ka onwards29, our data suggest that they inherited a resource that was already severely depleted. Analyses of pollen and charcoal in a drill-core spanning the past 27.5 kyr shows that fire had long been a part of the regional ecology prior to humans63. Early foragers probably used fire to convert forest and woodlands to grasslands64, but Paduano et al.63 argue that burning had no measurable effect on soil erosion until about 2600 cal. yr BP when humans had increased the vulnerability of upland soils by clearing natural vegetation, introducing domesticated camelids, and cultivating hillslopes for food crops such as quinoa43. Similarly, we hypothesize that in the Belén catchment, soil thinning was linked to deforestation, land clearance for agriculture and herding intensification in a context of climatic variability63.It is well established that soils become vulnerable to rainsplash and sheetwash erosion when subjected to fire, grazing or cultivation, which disrupt the capacity of plants to bind the soil. Although Andean societies of the last millennium continued to degrade soils45. A more persuasive argument is that Andean agropastoralist communities were responding to climate variability by modifying their environment in highly dynamic ways65. The catastrophic soil depletion documented here is therefore a result of the transformative effects on their landscape and is plausibly tied to the onset of the Early Anthropocene in the Andean Altiplano. Alongside the broadscale human-driven transformation of Earth’s surface recognized around 4–3 ka67, our findings support a growing challenge to the idea of a late Anthropocene tied solely to the industrial era12.Although we cannot separate the relative influence of environmental vs. anthropogenic forcing on long-term soil sustainability, we question whether such partitioning is even possible given the technological and social transformation unfolding in the Andes at the timen = 3), hilltop stony soil (n = 6) and river sand (n = 4). All hilltop samples were collected at the crests of hills to ensure sampling of in situ material not subject to downslope transport. Bedrock samples were collected from outcrops of Silurian sandstone (13-1), Devonian quartzite (01) and Cretaceous sandstone (13-7). A maximum sample thickness of 5 cm below the surface was taken. Soil samples comprise amalgamated clasts from Oligocene–Miocene conglomerates , matrix sand from Oligocene–Miocene conglomerate  and amalgamated siliceous chips from hardpan soils developed on Oligocene–Miocene siltstone . Siliceous hardpan is a pedogenic concentration of opaline silica commonly found in arid landscapes68. All rock and clast samples were crushed and sieved to a grain size of <0.6 mm. Quartz was isolated via leaching with hydrochloric acid (HCl) and weak hydrofluoric acid (HF).We collected a total of 13 samples including hilltop bedrock  × 10−15 (±1σ) was subtracted from the measured 10Be/9Be ratio. The total Al concentration was determined by inductively coupled plasma quadrupole mass spectrometry measurements at ETH Zürich on sample aliquots taken immediately after quartz dissolution. All results are summarized in the Supplementary Table About 22–30 g of each clean quartz sample was spiked with ~250–300 μg of 14C was extracted from aliquots (∼3–5 g) of the same purified quartz samples analysed for 10Be and 26Al. Samples 01, 07, 08, 09, 11 and 12 were analysed in 2012 at the ETH Zürich extraction system as outlined in ref. 70. Samples 13-1, 13-4, 13-5, 13-7, 15-2 and 15-20 were analysed in 2018 on a new extraction system at ETH Zürich71. In both systems, atmospheric 14C adsorbed to crystal surfaces is removed by sample preheating (~500 °C) followed by release of cosmogenic 14C from the quartz crystal lattice by high-temperature diffusion (~1600–1670 °C for 2–3 h). The CO2 samples were measured without prior graphitization at ETH Zürich MICADAS accelerator mass spectrometer system equipped with a gas ion source72. Data reduction followed the protocol of ref. 73 including the subtraction of a long-term process blank applicable to the time of extraction  production rates of 4.01 ± 0.33 at g−1 yr−1 for 10Be, 27.90 ± 2.80 at g−1 yr−1 for 26Al and 12.20 ± 0.89 at g−1 yr−1 for 14C76. Muonic production rates at the surface are based on muon interaction cross-sections for 10Be-26Al76 and 14C78. Depth-dependence of nuclide production was modelled via simple exponential functions with attenuation lengths 160, 1500 and 4320 g cm−2 for production via spallation, negative muon capture and fast muons, respectively. We assume a density of 2.65 g cm−3 for bedrock samples; for all other samples (clasts and river sand), we assume a density of 2.0 g cm−3. This is a valid approach also for the clasts, which have higher density but have been exhumed via removal of the lower-density matrix. For sample 09, we determined a 26Al concentration that yields a 26Al/10Be ratio of 3.1, which we consider to be implausibly low. This sample contains significantly lower total Al (∼7 p.p.m.) relative to other samples and 2.4-fold lower total Al than sample 15-20, which was collected from the same lithology at a nearby hilltop . The model parameters are  for the step model and  for the spike model where t is the time of erosion rate change or the erosion event (yr BP), respectively, and x is the depth of surface lowering (m). For each model run, 32 random walkers search the parameter space defined as ε1: 0.1–25 mm kyr−1, ε2/ε1: 0.1–1000, εconst: 0.05–30 mm kyr−1, x = 0–3 m and t: 1–10,000 yr. For the joint inversion, the time t is shared by all sample localities, but the erosion rates and sediment loss are allowed to vary from place to place. Each walker begins with a burn-in phase of 5000 iterations that allow for a coarse initial search of the model space. Using the results of the burn-in phase, a detailed search of the model parameters is performed with 40,000 iterations yielding 1.28 million iterations in total . Walkers aim for a 40% acceptance ratio. In addition to the joint inversion, we apply individual inversions that allow the time of change to vary between sampling localities (Supplementary Fig. We apply an MCMC-based inversion model14C in the hilltop samples might result from soil mixing, we employ a numerical model80 to investigate changes in 14C and 10Be concentrations within a variably mixed soil layer. Assuming that mixing diffusivity decreases exponentially with depth, diffusivity at depth z may be expressed as:m0 is the diffusivity at the surface and D the diffusivity decay depth. We consider that depth-dependent soil diffusivity better represents natural soil-mixing processes than the end-member case of steady erosion and complete uniform mixing68. To compare the modelled steady-state depth profiles for 14C and 10Be in a non-uniform mixed soil layer (Supplementary Fig. −1 according to the average 10Be-derived erosion rate from our study area, a surface diffusivity of 0.1 cm2 yr−1 and D values of 0, 20, 40, 60, 80, 100 and 120 cm. To accommodate the slight variation in nuclide production rates at the different sampling locations, we apply SLHL reference production rates for 10Be and 14C as given above and scale the measured concentrations accordingly. The deviation of the 14C concentration and the 10Be/14C ratio in a mixed soil layer from a steadily eroding non-mixed surface is calculated for surface erosion rates between 0.1 and 1000 mm kyr−1 using the same diffusivity parameters and production rates (Supplementary Fig. Unconsolidated sediments or soils can be affected by mixing processes due to bioturbation and/or cryoturbation. To assess whether the measured depletion of Supplementary Information


Bacillus subtilis is a soil-dwelling organism that can be isolated from a wide variety of environments. An interesting characteristic of this bacterium is its ability to form biofilms that display complex heterogeneity: individual, clonal cells develop diverse phenotypes in response to different environmental conditions within the biofilm. Here, we scrutinized the impact that the number and variety of the Rap-Phr family of regulators and cell-cell communication modules of B. subtilis has on genetic adaptation and evolution. We examine how the Rap family of phosphatase regulators impacts sporulation in diverse niches using a library of single and double rap-phr mutants in competition under 4 distinct growth conditions. Using specific DNA barcodes and whole-genome sequencing, population dynamics were followed, revealing the impact of individual Rap phosphatases and arising mutations on the adaptability of B. subtilis.Microbes commonly display great genetic plasticity, which has allowed them to colonize all ecological niches on Earth.  rap-phr genes, parts of peptide-based quorum sensing systems, impacts the sporulation and biofilm formation of B. subtilis under four different conditions and a multitude of strains. Using strain-specific barcoding and complete genome sequencing, the authors show that competitive selection is driven by acquired mutations, and that selected strains demonstrate increased spore fitness relative to their ancestors and wild types.Ramses Gallegos-Monterrosa, Mathilde Nordgaard Christensen, and colleagues investigate how a single or double deletion of the  Bacillus subtilis has become a model organism for the study of biofilms and population differentiation due to its ability to develop diverse phenotypes within an isogenic population2. Even when grown in liquid mixed cultures, where environmental conditions are assumed to be homogeneous, B. subtilis cells can be found as single flagellated cells or as non-flagellated chained cells due to stochastic variation in the expression of motility-related genes3. This population heterogeneity further increases when B. subtilis develops a biofilm, i.e., cells commit to particular functions, such as biofilm matrix production, exoenzyme secretion, or spore formation4. The development of these different cell types is partially triggered by the variation in the environmental conditions that exist in diverse parts of the biofilms, which can then be seen as a collection of ecological microniches, each with its own type of specialized inhabitant7.In recent years, B. subtilis possesses a complex regulatory network that leads the cells within the biofilm to generate a phenotypically heterogeneous population. This network is described to be mainly controlled by the master transcriptional regulators Spo0A, DegU, and ComA. The activity of these regulators depends on their phosphorylation status, which is controlled by the activity of specific kinases that can sense a wide array of environmental and intracellular signals, and phosphorylate their corresponding response regulators accordingly. DegU and ComA are phosphorylated by kinases DegS and ComP, respectively, while Spo0A can be activated by five different kinases that act through a phosphorelay formed by the response regulators Spo0F and Spo0B. Furthermore, the regulatory network includes multiple cross-talk mechanisms and regulatory feedback loops that contribute to its modulation by constantly monitoring the general metabolic state of each cell within the biofilm9.B. subtilis is further controlled by a family of response regulator aspartyl-phosphate (Rap) phosphatases and their cognate phosphatase-regulator (Phr) peptides. The cytoplasmic Rap proteins exert their regulatory function by inhibiting the activity of their target regulator  via dephosphorylation, or by directly blocking DNA binding. The Rap proteins are, in turn, inhibited by their cognate Phr peptides, which are produced as pre-Phr proteins that are exported to the extracellular milieu and cleaved to produce mature five to six amino acid Phr peptides. The Phr peptides are imported back into the cell upon reaching threshold concentrations at high cell density and bind to their cognate Rap phosphatase, inducing conformational changes that inhibit its activity11. The rap and phr genes are usually found as pairs in the same genetic loci, with the phr genes following and in some, but not all cases slightly overlapping the corresponding rap genes, and therefore the expression of these genes being transcriptionally coupled14. Furthermore, some of the rap genes are not followed by a corresponding phr gene. Additionally, some Rap proteins can be regulated by Phr peptides that are encoded in other cassettes, e.g., RapB and RapJ are controlled by PhrC15.The population-heterogeneity regulatory network of Bacillus genus, with ca. 2700 rap genes recently reported to be distributed among 346 Bacilli genomes; from those, ca. 80 different putative rap–phr alleles were found in the B. subtilis group alone16. Only a small minority of the B. subtilis Rap phosphatases has been characterized, finding that they have high redundancy in their regulatory function: most of them target Spo0F, ComA, or both; and only one (RapG) has been described to act on DegU11. Interestingly, B. subtilis shows great genomic plasticity regarding rap–phr gene pairs; 127 recently compared strains of the B. subtilis group were shown to have multiple rap–phr gene pairs, with an average of 11 rap genes per strain16. This genetic variation among B. subtilis strains is not superfluous: since the Rap phosphatases modulate the activity of the main regulators of population heterogeneity, it has been proposed that the Rap–Phr pairs serve to adjust this regulatory network of B. subtilis to the needs of particular ecological niches18. As an example, it has been shown that B. subtilis strains isolated from gastrointestinal tracts of diverse animals have diverse sporulation initiation rates, with some being able to start sporulating already during logarithmic growth phase. This variation is correlated to the presence or absence of specific Rap–Phr pairs, and thus it has been suggested that the precise combination of rap–phr gene pairs matches the particular sporulation needs of a given niche17.The Rap–Phr regulatory pairs are highly prevalent in the 10, bacterial social interactions and evolutionary dynamics have been suggested to influence the emergence and persistence of multiple Rap–Phr systems with redundant functions in B. subtilis16. This idea is supported by the fact that up to 75% of rap–phr gene pairs are located in sections of the genome related to mobile genetic elements (such as prophages and transposons), suggesting that Rap–Phr systems are commonly acquired by horizontal gene transfer among Bacillus strains16; moreover, at least one B. subtilis Rap phosphatase (RapI) is known to promote the propagation of the mobile genetic element that contains it19. The genetic variation in Rap–Phr systems among B. subtilis strains complicates the understanding of the role that the whole set of Rap phosphatases plays in modulating the population-heterogeneity regulatory network of any particular B. subtilis strain. Furthermore, the best-known Rap–Phr systems have usually been studied independently from each other, in diverse genetic backgrounds, and using different experimental conditions11. Likewise, previous investigations have focused on different aspects of Rap–Phr regulation, e.g.: RapA and RapB have been mainly studied for their role in sporulation regulation20, while RapC and RapF are known to regulate competence development22. Conspicuously, Rap–Phr systems have not been thoroughly investigated with regards to biofilm formation11; to the best of our knowledge, only RapP has been previously shown to affect their formation24.Since the Phr peptides function as quorum sensing molecules regulating the production of costly public and private goodsrap–phr mutants or combined double mutations. To address this complexity, we used a barcoding approach that allowed us to follow the relative abundance of 79 strains, wild-type, single, and double rap–phr deletion mutants, within the populations subjected to various selective conditions.Here, we aimed at revealing the influence of cultivation time (2 versus 5 days) and condition (planktonic versus biofilm) on selection toward particular B. subtilis, particularly on sporulation within biofilms, and how absence or presence of different Rap phosphatases would affect the adaptability of B. subtilis to different growth conditions. We used B. subtilis DK1042 (hereafter WT), which is a transformable strain derived from the isolate NCIB 3610 harboring a comIQ12I mutation25. Strains NCIB 3610 and DK1042 have not undergone the domestication process that other commonly used B. subtilis strains have. This domestication can lead to loss of genetic functions and regulation changes, including modifications to Rap–Phr systems27. The WT has 11 rap genes encoded in its genome (rapA–rapK), additionally, it also possesses one more Rap–Phr system (rapP–phrP) encoded in its pBS32 plasmid23. We created single knockout mutant strains of all the rap–phr genes, and double knockout mutant strains that lack two rap–phr gene pairs in all possible combinations. In all cases both the rap and phr genes were deleted from the genome or plasmid. All created strains and the WT were further tagged with a pre-identified specific DNA barcode: a randomly generated 12 bp nucleotide sequence that was integrated into the amyE locus of each strain. We used an experimental competition approach to analyze how the different Rap–Phr system combinations would impact the adaptability of all mutant strains (and WT as control) to two different growth conditions: shaken liquid cultures, where cells would multiply in a planktonic state; or static liquid cultures, where cells would form a pellicle biofilm on the air-liquid interface  or microplate wells (for spores obtained from pellicle cultures) that were then incubated under the same conditions. The competition experiment was maintained for a total of nine reinoculation cycles for each culture condition. Full details of the competition experiment can be found in the “Methods.” We purified total DNA from the cultures obtained after the first, third, fifth, seventh, and ninth culture cycle, and from the mix used to initiate the competition. The DNA was used to PCR amplify the amyE locus containing the strain-specific DNA barcodes. Using high-throughput sequencing, we were able to dissect the population dynamics of all the used strains throughout the competition experiment by analyzing their representation ratios within the competing population.We were interested in systematically determining the impact that each Rap system Fig. a has on ace Fig. . All stun = 11 for 2-day planktonic, n = 24 for the 5-day planktonic, n = 17 for the 2-day pellicle, and n = 24 for the 5-day pellicle conditions). Importantly, no correlation was detected in the variation of each strain between the initial inoculation mixtures and after the ninth transfer in each selective condition , whilst mix, in contrast, had none . Overall, this suggests that the major contributor to the observed variation is the genotype rather than the mix. One particular mutant (ΔrapEΔrapJ) was strongly selected after nine transfers; however, only in mixture D, which suggests possible preexisting mutation in the overnight grown culture benefiting this particular strain at the time of inoculation. However, this mutant was not increased similarly in other mixtures. Interestingly, strains that slightly increased their relative abundance in the population after the first culture transfer did not necessarily maintain this trend throughout the competition experiment, in both planktonic- and pellicle-forming conditions, e.g., ΔrapAΔrapD or ΔrapAΔrapF mutants  and the biofilm matrix regulator sinR (L74S in 2-day pellicle culture) genes, the comA gene contained a frame shift mutation in one of the isolates from 2-day pellicle culture. In addition, mutations were identified in genes related to sporulation and germination, including gerKA (3 isolates), gerKC (2 isolates), gerAB (1 isolate), and also in the noncoding region upstream of yhaX, a gene that codes for a SigE-dependent sporulation protein. Finally, the pBS32 plasmid, therefore also the rapP–phrP genes, was lost in one of the 5 days cultured pellicle isolates that contained rapB, rapD–phrD deletions. None of the deleted rap–phr modules were restored in any of the isolates . Additional mutations were also detected unrelated to sporulation or with unknown function , gerAB  isolate,29. Indeed, testing the culture supernatants of these evolved isolates against the indicator strain showed the presence of plaque formation , increases in copy number in various 3610 or domesticated 168 derivatives of B. subtilis when a sporulation bottleneck is used for selection. However, the direct role of the Rap-homolog in phi3T on sporulation or production of high-quality spores that germinate faster is unknown29, as is the target of RapX. However, we hypothesized that in addition to such a phi3T-driven genetic adaptation that easily spreads around in the whole population, selection might be still driven by certain rap–phr deletions.Importantly, nine out of the ten isolated evolved strains spontaneously released phage particles that showed lytic activity toward the ancestral strain , two different competition experiments were performed. To test the impact of the original rap–phr mutations on fitness and sporulation, some of the ancestor ∆rap mutant strains were competed independently against the WT  were plated for CFU counting, and the relative fitness  and sporulation fitness (SF) of ancestor versus WT, and of evolved versus ancestor was calculated .To test the hypothesis of the population dynamics being affected both by the original  WT Fig. . The com WT Fig. . Further∆rapB, ∆rapB∆rapD, ∆rapB∆rapE, ∆rapB∆rapH, ∆rapC, ∆rapE∆rapF, ∆rapE∆rapH, ∆rapH∆rapI, and ∆rapH∆rapP ancestors showed significantly increased fitness and/or SF compared to the WT  show strains ∆rapD∆rapJ and ∆rapH∆rapJ to increase their population abundance, but this is not the case for the populations grown under pellicle-forming conditions for 5 days. On the other hand, mutants in specific Rap–Phr systems do show consistently decreased population abundance across all tested conditions, e.g., most double rap–phr mutants with a ∆rapK deletion. Our competition approach allows us to compare the effect of particular rap–phr mutations across different conditions, and thus analyze their regulatory function: RapK has been previously suggested to inhibit ComA activity19, here we show that various ∆rapK strains when harboring other rap deletions  suffer a loss of competition fitness when selecting for spores. We hypothesize that the ∆rapK mutation leads to an overexpression of ComA-controlled genes related to production of antimicrobials30, exoenzymes31, and competence development32, this, in turn, may direct metabolic resources away from spore production and maturation, thus resulting in the observed loss of competition fitness, but only in combination with other ∆rap mutation. Another interesting behavior was that rap–phr mutants that increased their population representation after the first transfer did not necessarily maintain their abundance throughout the multi-strain competition experiment . However, of those only the ∆rapB∆rapE, ∆rapF∆rapI, ∆rapG∆rapI, and ∆rapH∆rapI strains kept this trend throughout the experiment and showed increased population representation in most replicates after transfer 9. On the other hand, ∆rapD∆rapJ, ∆rapF∆rapJ, ∆rapH∆rapJ, ∆rapJ, and ∆rapJ∆rapK mutants did not show increased abundance after transfer 1, but were increased after transfer 9. This highlights the versatility that the regulatory effect of Rap–Phr systems confers to B. subtilis’ ability to generate a phenotypically diverse population. Certain rap–phr mutant strains display an initially increased fitness when competing under conditions where all strains are present in the same ratio (starting conditions of the competition), while other strains show an increase in abundance over time. Their particular rap–phr mutations allows them to capitalize on hypothetically small fitness advantages when producing mature spores. Furthermore, during the sequential competition transfers, evolutionary adaptation to the experimental conditions of other strains in the population may take place, resulting in those strains gaining an increased SF, and possibly taking over the strains initially showing an increase in population abundance. In support of this, resequencing of ten clones isolated from random populations at the end of the competition experiment revealed that eight of the ten strains had gained mutations in different genes, including genes involved in the population-heterogeneity regulatory network, such as sporulation. This indicates that the strains can quickly acquire additional mutations that help them further adapt to the growth-sporulation cycles applied during the competition experiment.Our initial hypothesis was that the strains initially showing increased population representation in the first culture and subsequent sporulation would be the ones adapting most efficiently to the growth-sporulation cycles applied during our competition, and thereby be the ones winning (having a large increase in their population proportion) at the end of the experiment. Interestingly, no single strain was detected to be a transcendent winner for all of the four different conditions at the end of the competition experiment. Instead, various rap–phr mutants with WT and the evolved rap–phr mutants with their respective ancestors. For example, the ∆rapB∆rapE ancestor showed enhanced fitness and SF compared to the WT after 2-day pellicle formation, while the evolved strain isolated from a 2-day pellicle culture at the end of the competition experiment (transfer 9) showed increased pellicle but reduced spore fitness compared to the ancestor. This evolved ∆rapB∆rapE strain had gained mutations in two genes, gerKC and yqcG encoding a membrane receptor involved in spore germination33 and a toxin34, respectively. The ∆rapB∆rapE mutant that maintained the increased abundance in the 2-day pellicle conditions throughout the nine transfers is therefore presumably a result of both deletion in ∆rapB and ∆rapE, as well as the mutations arising during the experiment. Another rap–phr mutants with positive fitness or spore fitness compared to the ancestor WT also acquired mutations that may have increased their fitness during the mutli-strain competition experiment, e.g., mutation in degS in the ∆rapH∆rapI background. The observation that the majority of the isolated evolved strains show enhanced SF compared to the cognate ancestor indicates that the mutations arising during the experiment and the acquired awakening of the phi3T phage have a dramatic impact on the SF, thereby allowing the strains to adapt to the growth-sporulation cycles applied in the competition experiment. This, as well as the observation that most of the rap mutations have an effect on fitness and sporulation, indicates that the population dynamics during the experiment are a result of both the original rap–phr mutations present in the 78 mutant strains, as well as the additional mutations and phi3T arising during the experiment.Competition experiments quantifying fitness and spore fitness were employed to compare rap–phr strains between 2- and 5-day culture conditions, and between planktonic- and pellicle-forming conditions points to dissimilar selection pressures generated by these growth conditions that further reveal the role that Rap–Phr systems play in environmental adaptability, focusing on the ability of cells to generate mature spores, and to form pellicles. Additionally, genetic drift and negative frequency-dependent selection may occur under the used conditions.The differences in the variance of surviving B. subtilis strains strongly impacts their ability to compete in diverse environments, and that these systems are particularly important if adaptation depends on differentiation of particular cell types such as spores. We propose that the diversity of Rap–Phr regulatory systems allows B. subtilis to fine-tune its genetic regulatory network in order to quickly adapt to new ecological niches, possibly accompanied by the acquisition of additional mutations that provide the bacterium with increased fitness. Furthermore, genetic exchange of elements of a family of regulatory proteins could be a general mechanism for genetically related bacteria to more efficiently and quickly adapt to new environments.Our results indicate that variability in Rap–Phr systems among −1 tryptone, 5 g L−1 yeast extract, and 5 g L−1 NaCl) at 37 °C and shaken at 225 rpm. LB medium was used for all B. subtilis and Escherichia coli transformations. MSgg medium (5 mM potassium phosphates buffer (pH 7), 100 mM MOPS, 2 mM MgCl2, 700 µM CaCl2, 100 µM MnCl2, 50 µM FeCl3, 1 µM ZnCl2, 2 µM thiamine, 0.5% glycerol, 0.5% glutamate, 50 µM L-tryptophan, and 50 µM L-phenylalanine, adapted from35) was used for the competition experiment and to examine growth kinetics and pellicle formation. GCHE medium , 3 mM trisodium citrate, 3 mM MgSO4, 22 mg L−1 ferric ammonium citrate, 50 mg L−1 L-tryptophan, and 0.1% casein hydrolysate) was used to induce natural competence in B. subtilis36. Gallegos Rich medium was used to grow Lactococcus lactis MG1363, in order to purify pMH66: 21 g L−1 tryptone, 5 g L−1 yeast extract, 8.3 g L−1 NaCl, 3 g L−1 soya peptone, 2.6 g L−1 glucose, and 2.5 g L−1 MgSO4∙7H2O37. Media were supplemented with Bacto agar 1.5 % when media were needed for preparing plates. Antibiotics were used at the following final concentrations: kanamycin, 10 µg mL−1; chloramphenicol (chl), 5 µg mL−1; erythromycin-lincomycin (MLS), 0.5 µg mL−1 and 12.5 µg mL−1, respectively; ampicillin, 100 µg mL−1; spectinomycin, 100 µg mL−1; tetracycline, 10 µg mL−1.When fresh cultures were needed, strains were pre-grown overnight in Lysogeny broth (LB) medium -derived plasmids containing an antibiotic resistance cassette  flanked by Cre recombinase recognition sites lox66 and lox71. The respective antibiotic resistance genes have been amplified using oligonucleotides indicated in Supplementary Data 38, pWK-Sp (spectinomycin)39, and pDR183 (MLS)40 as templates. Thus, the obtained plasmids contain an antibiotic resistance cassette flanked by the upstream and downstream regions of a given rap–phr gene pair. All plasmids were created and maintained in E. coli MC1061.All strains used in this study are listed in Supplementary Data B. subtilis mutants of a single rap–phr pair were created via transformation of DK1042 with the corresponding plasmid containing the flanking regions of the target rap–phr pair. Double rap–phr mutants were created by transforming clean-deletion mutants of single rap–phr pairs with genomic DNA of strains that had the desired rap–phr mutation still with the corresponding antibiotic resistance cassette. All B. subtilis strains generated in this work were obtained via natural competence transformation36. Briefly, overnight cultures of the receiver strains grown in LB medium were diluted to a 1:50 ratio with GCHE medium, these cultures were incubated at 37 °C for 4 h with shaking at 225 rpm. After this incubation period, 5–10 µg of genomic or plasmid DNA was mixed with 500 µL of competent cells and further incubated for 2 h before plating on LB plates added with selection antibiotics. B. subtilis clean-deletion mutants of single rap–phr gene pairs were obtained by using the Cre recombinase expressed from plasmid pMH66 to eliminate their corresponding antibiotic resistance cassette, and subsequently curing pMH66 via thermal elimination of the plasmid41. Briefly, strains were transformed with 10 µg of pMH66, selecting transformants via incubation at 37 °C on LB plates added with tetracycline. Candidates were then screened for their capacity to grow at 37 °C on LB plates added with the antibiotic to which their parental strains (prior to transformation with pMH66) were resistant, those that were not able to grow were further incubated on LB plates at 43 °C for 18 h to induce the loss of pMH66. Candidates that were then unable to grow at 37 °C on LB plates added with tetracycline were considered to have lost pMH66.B. subtilis DK1042 and all single and double rap–phr mutants were marked with a randomly generated DNA 12 bp barcode in their amyE locus. To do this, plasmid pTB666 was created by cloning a chl resistance cassette (cat) into pTB1642, substituting its original kanamycin resistance cassette. The cat cassette was amplified from pNW33n using primers oTB118 and oTB119  at 600 nm to 1.0. Each starting mix was used to inoculate 100 ml bottles (0.5 ml of mix + 9.5 ml of MSgg medium) and 2 ml microplate wells (100 µl of mix + 1900 µl of MSgg medium). The experimental competition used four growth conditions: planktonic growth (10 ml culture in bottles shaken at 200 rpm) or pellicle development (static 2 ml in 24-well microplate), and incubation for 2 or 5 days. Twenty-four replicate populations were used for each growth condition (six replicates from each starting mix). All cultures were incubated at 30 °C throughout the experiment.The experimental competition was done using the barcoded versions of −1); step 3, extended the incubation time to 25 min; step 6, extended the incubation time to 45 min.After each incubation period, spores obtained from each population replicate were used to inoculate the next iteration of the same population. For this, pellicles obtained from the microplate cultures were collected in Eppendorf tubes with 1 ml of MSgg medium and sonicated until the pellicles were completely dispersed; afterwards, 100 µl aliquots from the dispersed pellicles, or 500 µl aliquots from the planktonic culture bottles were incubated at 80 °C for 20 min in order to kill all vegetative cells. After the incubation period, the heat-treated aliquots were used to inoculate new 100 ml bottles or 2 ml microplate well using the same volumes as during the initiation of the experiment. This regime was followed during nine culture reinoculation cycles, resulting in >39 generations. Additionally, DNA was obtained from aliquots of the starter mixes and from aliquots of each population replicate obtained before the heat treatment during the first, third, fifth, seventh, and ninth culture reinoculations. The DNA extraction was performed with the GeneMatrix Bacterial and Yeast Genomic DNA Purification Kit  with the following modifications to the manufacturer’s instructions: step 2, added 10 µl of lysozyme (10 mg mlB. subtilis amyE locus containing the barcodes was PCR amplified from the DNA samples obtained from the competition experiment using primers oBC1–oBC4 and oBC5–oBC16. These primers contain distinct 5-bp (for oBC1–4 primers) or 7-bp (for oBC5–16 primers) sequences that allowed us to identify up to 48 individual replicate populations per Illumina sequencing run. Data analysis was carried out using the R statistics environment44. PCR products, each represented by one R1-R2 Illumina sequence pair, were looked up for the presence of the 79 barcodes that differentiate between the 79 bacterial strains used in the study .We examined the ability of all barcoded strains to grow on MSgg medium in order to assess the impact of the 600 0.1. 20 µl of the O.D.-adjusted cultures was added to 2 ml of MSgg liquid medium in 2 ml microplate wells. These cultures were incubated at 30 °C for 5 days. After 2 days of incubation and at the end of the incubation period the obtained pellicles were examined with an Axio Zoom V16 stereomicroscope  equipped with a Zeiss CL 9000 LED light source, a PlanApo Z ×0.5 objective, and AxioCam MRm monochrome camera .We examined the ability of all barcoded strains to form pellicles on MSgg medium. Overnight LB liquid cultures of all barcoded strains were adjusted to O.D.® Ultra™ II DNA Library Prep Kit for Illumina. Paired-end fragment reads were generated on an Illumina NextSeq sequencer using TG NextSeq® 500/550 High Output Kit v2 (300 cycles). Primary data analysis  was carried out with “bcl2fastq” software . All further analysis steps were done in CLC Genomics Workbench Tool 9.5.1. Reads were quality-trimmed using an error probability of 0.05 (Q13) as the threshold. In addition, the first ten bases of each read were removed. Reads that displayed ≥ 80% similarity to the reference over ≥ 80% of their read lengths were used in mapping. Nonspecific reads were randomly placed to one of their possible genomic locations. Quality-based SNP (single nucleotide polymorphism) and small In/Del variant calling was carried out requiring ≥8× read coverage with ≥25% variant frequency. Only variants supported by good quality bases (Q ≥ 20) were considered and only when they were supported by evidence from both DNA strands.Ten populations from the end of the competition experiment were selected randomly (representing at least two replicates of each growth condition). Aliquots of the selected populations were used to inoculate LB plates that were incubated overnight at 30 °C in order to isolate clones from each population. Overnight LB liquid cultures of the isolated clones were used to extract genomic DNA using the GeneMatrix Bacterial and Yeast Genomic DNA Purification Kit . Paired-end libraries were prepared using the NEBNext600. The WT strain was labeled with mKATE to facilitate counting of WT colonies on agar plates after the competition experiments. None-barcoded ancestors were competed against WT, whereas the evolved, barcoded strains, isolated after transfer 9, were mixed with their cognate (none-barcoded) ancestor strain in a 1:1 ratio. From this mix, 15 µL was added to 1.5 mL MSgg liquid medium in 2 mL microplate wells in a 24-well plate. The cultures were grown under static conditions at 30 °C for 48 h. After incubation, the pellicles were harvested and transferred into Eppendorf tubes containing 1 mL NaCl 0.9% (Carl Roth) and subjected to standard sonication protocol allowing proper disruption of the biofilm without damaging the cells. Afterwards, 200 µL of the obtained cell suspensions was incubated at 80 °C for 20 min, killing all vegetative cells leaving only spores left. CFU assays were performed for cell suspensions  and heat-treated aliquots (spores only). For the ancestor versus WT competition, obtained cell suspensions were plated on LB plates for CFU counting. For the evolved versus ancestor competition assays, cell suspensions were plated on two different types of plates: LB plates and LB + chl (5 µg ml−1). LB medium allowed growth of both evolved and ancestors, while LB + chl only allows growth of evolved strains due to the presence of a chl resistance gene next to the barcode gene in the evolved strains. Thereby, CFU of evolved strains equals the CFU obtained from the LB + chl plates, while CFU of ancestors equals the CFU on the LB plates (evolved + ancestors) subtracted the CFU of LB + chl plates (evolved only).For competition experiments in pellicles, overnight LB liquid cultures of ancestors, evolved strains, and WT labeled with mKATE were adjusted to same ODA, WA, in competition with strain B was calculated as follows:Relative fitness was calculated for ancestor strains relative to the mKATE-labeled WT, and for evolved strains relative to their cognate ancestors. Relative fitness for strain A (SFA) in competition with strain B was calculated as follows:In addition, SF of ancestor strains relative to WT and of evolved strains relative to ancestors was calculated. Relative SF for strain W) = 0  were calculated using one-sample t-test with test mean = 0 and a significance level of 0.05. Prior to using t tests, normality was tested by Shapiro–Wilks test. PCA on aggregated and scaled data was used to visualize the multivariate pattern of each genotype stratified by mix. Analogously, PERMANOVA, using Euclidian distances, was used to test the statistical effect of both these variables. No statistical tests were used to determine sample sizes. Such was chosen based on experience and knowledge on expected biological variability (which cannot be controlled by the experimenter) and accuracy of the experimental methods used. No data were excluded from the analyses. All experiments contained biological replicates with specific information on replicates in the “Methods,” and the vast majority of the experiments were performed multiple times.Statistical analysis was performed using OriginPro 2018b . The relative fitness values calculated from the competition experiments were log-transformed, and statistical differences from log10(epsΔtasA). Biofilm mutant was used as a lawn to improve the visibility of plaques. Phage particles were purified like described previously46. Specifically, culture supernatants were collected, mixed at a 1:4 ratio with PEG-8000 solution , incubated on ice for at least 90 min and precipitated by centrifugation . The pellet was resuspended in 10% of the original supernatant volume in TBS solution , incubated on ice for 90 min and centrifuged . Supernatant was carefully transferred to clean Eppendorf tubes and phages were visualized using electron microscopy.Supernatants, obtained from overnight cultures of the evolved strains, were spotted on soft-agar (0.3%) lawn of the ancestor strain with knockout of biofilm genes (NCBI 3610 ΔBefore use, 400 mesh nickel grids with a 3–4 nm-thick carbon film, CF400-Ni-UL EMS Diasum, were hydrophilized by 30 s of electric glow discharging. Next, 5 μl of purified phage solutions were applied onto the grids and allowed to adsorb for 1 min. The grids were rinsed three times on droplets of milliQ water and subjected to staining with 2% uranyl acetate. Specifically, with a help of EM grid-grade tweezers, the grids were placed sequentially on droplets of 2% uranyl acetate solution for 10, 2, and 20 s. Excess uranyl acetate was wicked away using filter paper and the grids were allowed to dry overnight and stored in a desiccator until analysis. Transmission electron microscopy was performed utilizing a FEI Tecnai T12 Biotwin TEM operating at 120 kV located at the Center for Electron Nanoscopy at the Technical University of Denmark, and images were acquired using a Bottom-mounted CCD, Gatan Orius SC1000WC.Further information on research design is available in the Supplementary InformationSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Description of Additional Supplementary FilesPeer Review FileReporting Summary


Oxidative stress is an important factor involved in the progress of heart failure. The current study was performed to investigate whether pinocembrin was able to ameliorate post-infarct heart failure (PIHF) and the underlying mechanisms.Rats were carried out left anterior descending artery ligation to induce myocardial infarction and subsequently raised for 6 weeks to produce chronic heart failure. Then pinocembrin was administrated every other day for 2 weeks. The effects were evaluated by echocardiography, western blot, Masson’s staining, biochemical examinations, immunohistochemistry, and fluorescence. In vitro we also cultured H9c2 cardiomyocytes and cardiac myofibroblasts to further testify the mechanisms.We found that PIHF-induced deteriorations of cardiac functions were significantly ameliorated by administrating pinocembrin. In addition, the pinocembrin treatment also attenuated collagen deposition and augmented vascular endothelial growth factor receptor 2 in infarct border zone along with an attenuated apoptosis, which were related to an amelioration of oxidative stress evidenced by reduction of reactive oxygen species (ROS) in heart tissue and malondialdehyde (MDA) in serum, and increase of superoxide dismutase (SOD). This were accompanied by upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2)/ heme oxygenase-1 (HO-1) pathway. In vitro experiments we found that specific Nrf2 inhibitor significantly reversed the effects resulted from pinocembrin including antioxidant, anti-apoptosis, anti-fibrosis and neovascularization, which further indicated the amelioration of PIHF by pinocembrin was in a Nrf2/HO-1 pathway-dependent manner.Pinocembrin ameliorated cardiac functions and remodeling resulted from PIHF by ROS scavenging and Nrf2/HO-1 pathway activation which further attenuated collagen fibers deposition and apoptosis, and facilitated angiogenesis.The online version contains supplementary material available at 10.1186/s10020-021-00363-7. Heart failure (HF), characterized by a range of terminal clinical syndromes resulted from structural and/or functional impairments of heart, remains a global healthy burden, with myocardial infarction (MI) as the leading cause (Ziaeian and Fonarow Accumulating studies have shown that oxidative stress (OS) is predominantly involved in the pathological progression of PIHF  .56 male Sprague–Dawley rats, purchased from animal experiment center of China Three Gorges University, were raised in Wuhan No.3 Hospital animal administration. All experiments were officially permitted and performed in Hubei Key Laboratory of Cardiology, complied with the The rats initially received left anterior descending coronary artery (LAD) ligation or sham surgery. Briefly, rats were anesthetized by 3% pentobarbital , followed by tracheal intubation and respiratory support . Subsequent heart exposure was performed and the LAD was ligated by 7-0 monofilament nylon sutures. Occurrence of paleness of the apex cordis or ST segment elevation on II lead of electrocardiogram indicated the induction of MI. The sham operation underwent the identical procedures except the LAD ligation. Penicillin  was injected intramuscularly once a day for a week after operation.Afterwards, the rats were carefully raised for 6 weeks to produce PIHF. Rats were then randomly split into 4 groups: (i) sham + saline (sham group); (ii) sham + Pino group; (iii) LAD ligation + saline (HF group); (iv) LAD ligation + Pino (HF + Pino group). Pinocembrin  or same volume of saline was injected intravenously through the tail vein every other day for 2 weeks. The dosage referred to previous study ; LAD ligation + saline (HF group); LAD ligation + enalapril (HF + ACEI group). The rats underwent corresponding operation except that the rats of HF + ACEI group were fed orally by 20 mg/kg enalapril.The cardiac functions were evaluated by transthoracic echocardiography utilizing an ultrasound imaging system , as previously reported in our group -containing vacuum blood-collecting tubes with successive centrifugation at speed of 3000 r/min for 15 min and at a temperature of 4 °C, followed by isolation of serum supernatant and preservation at − 80 °C. Then the hearts were apace harvested. Part of infarct border zone of each heart was cut off and frozen in liquid nitrogen for western blot analysis, with the other heart tissue fixed in 4% paraformaldehyde for more than 24 h.We performed Masson’s staining to detect the collagen deposition in the infarct border zone. Paraformaldehyde-fixed ventricles were embedded by paraffin and then cut into 5-μm sections. The sections were subsequently stained with Masson trichrome where the collagen was dyed in blue, the myocardium in pink and the nucleus in black. The collagen deposition proportion was calculated by Image J software  at × 200 magnification.2O2 to block endogenous peroxidase, with incubation of 1 μg/mL VEGFR2 antibody  for 12 h at 4 ℃ fulfilled. Whereafter, the sections were incubated by horseradish peroxidase (HRP)-labeled secondary antibody  for 50 min at 37℃. In addition, 3,3’-diaminobenzidine was utilized to develop positive staining which was shown in tawny. Finally, the average optical density of VEGFR2 was analyzed and calculated by Image Pro Plus 6.0 software .Immunohistochemical analysis was performed to evaluate the density of vascular endothelial growth factor receptor 2 (VEGFR2) whose upregulation promoted angiogenesis that was salutary and important in attenuation of remodeling . Briefly, the ventricular sections were exposed to TUNEL reagent, where TdT and dUTP were mixed with a proportion of 1:9, for 1 h at room temperature, after incubation of proteinase K and rupture of cell membrane. Finally, the fluorescent images were observed at a wave length of 530 nm which was triggered by an emission wave length of 450 nm (× 400 magnification). The apoptotic rate of ventricular tissues in each group was analyzed and calculated by the Image J software.We implement western blot to elucidate the underlying mechanisms by which Pino ameliorated PIHF. The procedures of protein sample extraction and preparation resembled those previously reported in our study  subsequently substituted for normal medium in 3 wells to incubate the cells for 48 h to mimic HF state since HF is also characterized by an overactivation of sympathetic tone and stimulation of β1-adrenergic receptor generates ROS , were cultured to further verify the effects exerted by Pino in vitro. H9c2 cardiomyocytes were cultured in e Dulbecco's modified Eagle's medium (DMEM)/ F12 medium that contained 10% fetal bovine serum  under a temperature at 37 ℃ and an atmosphere of 95% air and 5% CO5 density in each well, by exploiting microplate reader at a wave length of 450 nm, where a higher value indicated an augmented proliferation activity.Neonatal rat cardiac myofibroblasts (CMFs) were cultured to investigate the effects of Pino on collagen secretion. The CMFs were separated by using neonatal rats of 3 days old, referring to previously demonstrated methods . DHE, which was dissolved in DMSO, was diluted to 5 μM for frozen sections or added to the serum medium to be diluted to the same concentration in accordance with the instructions. The paraformaldehyde-fixed hearts were frozen followed by being sliced up to 4-μm sections. The sections or H9c2 cardiomyocytes were co-incubated with DHE solution for 30 min at a temperature of 37 ℃, imaged afterwards by an inverted fluorescent microscope . Briefly, the nucleus which were filled with DNA and RNA were dyed in distinct red due to an affinity between nucleic acid and DHE. In addition, a stronger fluorescence intensity indicated more accumulation of ROS.Biochemical examinations were performed to probe the alterations of serum BNP, SOD and malondialdehyde (MDA) in serum and cellular supernatant which indicated HF phenotype and the oxidant defense, respectively. The concentration was measured by using corresponding biochemical assay kits according to manufacturer’s instructions .The continuous variables were represented as mean ± standard error while the proportional variables were shown as percentage. Comparisons in all groups were performed by using one-way analysis of variance (ANOVA) corrected by a Tukey post hoc test except those in echocardiography data which was analyzed by two-way ANOVA corrected by the Fisher least significant difference test. A p value < 0.5 was defined as statistically significance.We firstly implemented echocardiography to determine the HF phenotype establishment induced by LAD ligation and the effects exerted by Pino on cardiac functions. Representative two-dimension M-mode echocardiograms in all groups were illustrated in Fig. Given that the regenerative capacity of cardiomyocytes was sparse or absent, the improvements of cardiac functions were attributed more to amelioration of the alive cardiomyocytes and tissues by Pino rather than cardiomyocytes regeneration. Thus, we further evaluated 3 key factors in PIHF remodeling: fibrosis, neovascularization and apoptosis, as previously suggested  upon stresses/stimuli, and translocates to nucleus to bind to the promoter region of antioxidant response element (ARE), facilitating expression of a plethora of antioxidant genes and enzymes, among which the HO-1 was the representative enzyme and has a vital role  significant antioxidant efficacy that has been testified in previous studies and our present research; (ii) Pino could be extracted from a number of natural plants, honey and propolis or be artificially synthesized which ensured the sufficiency for production and a relative cheap price; (iii) richness of Pino in honey and propolis indicated a dietary uptake for convenience. Actually, a recent phase-I clinical trial has been performed to provide Pino for healthy subjects where Pino showed well tolerance and safety  Representative Masson staining images demonstrated collagen deposition in infarct border zone, with the infarct zone marked by arrows (black). (B) Fibrosis quantification in all groups. **: p < 0.01.