Pennisetum glaucum) and direct insights into human dietary reliance remain absent. Here, we utilise stable isotope analysis  of human and animal remains and charred food remains, as well as plant microparticles from dental calculus, to assess the importance of incoming crops in the Congo Basin. Our data, spanning the early Iron Age to recent history, reveals variation in the adoption of cereals, with a persistent focus on forest and freshwater resources in some areas. These data provide new dietary evidence and document the longevity of mosaic subsistence strategies in the region.The emergence of agriculture in Central Africa has previously been associated with the migration of Bantu-speaking populations during an anthropogenic or climate-driven ‘opening’ of the rainforest. However, such models are based on assumptions of environmental requirements of key crops (e.g.  Bleasdale et al. examine the introduction of agricultural crops in the Congo Basin with stable isotope analysis of human and animal remains, charred food remains, and plant microparticles from dental calculus. Their findings reveal variation in the adoption of cereals from the early Iron Age, and provide long-term insights into changing human reliance on different resources. While there has been an increasing departure from notions of a single sweeping ‘Bantu Expansion’, the degree to which the movement of people, languages, and the emergence of farming are linked across Africa continues to be forcefully debated7. Central Africa is at a key location for developing existing models for the spread of farming8 yet investigations of the emergence of food production, particularly in the rainforest, have been limited9. Assumptions that tropical rainforests represent substantial barriers to agriculturalists10 have been used to rationalise a relatively late arrival of farming in the region, c. 2500 years ago, during a period of climate- or human-induced deforestation13  can be grown in forested portions of the Inner Congo Basin27. This suggests that discoveries of pearl millet (c. 2330–330 BP) at Iron Age sites across Central Africa, regions presently covered in tropical rainforest28, need not represent a time of mass ‘rainforest crisis’30. Not only that, but Iron Age expansions into the various tributaries of the Congo River continued well after the supposed peak in rainforest decline 2500 years ago, suggesting more complex, ongoing processes of agricultural adaptation, and settlement. Together, these developments make it essential to build more integrated, multidisciplinary, and context-specific insights into changes in diet and land use in different parts of Central Africa through time, as different agricultural populations negotiated their tropical surroundings.Linguistic, material culture, and radiocarbon analyses have now shown that human arrival throughout the Congo Basin was a complex and time-transgressive occurrence, potentially with the interaction of different populations occurring13C), nitrogen (δ15N), and oxygen (δ18O) isotope analysis of human and animal remains. Isotopic results were obtained for human burials from the sites of Imbonga , Longa , Bolondo , and Matangai Turu Northwest . In addition, bone collagen (n = 10) and enamel (n = 6) were analysed for a range of fauna from BLD to create an isotopic baseline. The sites studied represent different geographic and temporal contexts  using the stable carbon δ3C, nitroxts Fig. , would h13C analysis of human tissues has long been demonstrated to provide insights into reliance on plants with different photosynthetic pathways (namely C4 versus C3) and their animal consumers , forest plants are C3, while incoming cereal crops  are C4. δ13C measurements of wild plants and animals from the rainforests of the DRC show that these forests are largely composed of C3 vegetation35. Moreover, they show a recognisable ‘canopy effect’ on this C3 vegetation that results in lower δ13C among plants, and their animal consumers, living under dense canopies compared to those living in more open areas35, something that has been well-documented in many other tropical regions36.δ3-dominated evergreen and semi-deciduous forest37. Stable carbon measurements of faunal tooth enamel from BLD, which largely reflect the proportions of C3/C4 plants consumed, reflect local palaeoecology, as well as providing baseline values for human diet. The final site, MTNW, is situated in the closed-canopy forest of the Ituri Region of the Northeast Congo Basin with palaeoenviromental proxies, suggesting a predominance of tropical forest tree taxa during the time of occupation39.The sites of IMB, LON, and BLD are located on tributaries of the Congo River in the western DRC Fig. an area 15N analysis provides insights into the positions of humans within their trophic web and their potential consumption of aquatic resources41, while δ18O measurements reflect water sources and environments43 . Two results generated for human burials from BLD were excluded from final analysis as they produced a %N < 5 and a %C < 15 % , domesticated browsers , mammalian carnivores (n = 2), and aquatic species . δ13C values from BLD mammals (n = 6) are consistent with a largely C3-based diet with measurements ranging from −23.7 to −18.2‰, although the goats, the dog, and the fox-sized carnivore could potentially have some C4 component to the diet  and small antelope (BLD 83/2-6 + 7) have δ15N values  consistent with herbivorous diets. In contrast, the fox-sized carnivore (13.7‰) and dog (12.3‰) display higher δ15N that is consistent with consumption of animal protein. Three of the aquatic species sampled had higher δ15N values than the catfish (δ15N 9.4‰), the crocodiles gave δ15N of 11.1 and 11.4‰, and the bichir (Polypterus sp.), a fish known to consume other fish and small vertebrates48, produced a δ15N measurement of 12.7‰. The accompanying δ13C value of −24.7‰ for the bichir somewhat overlaps with that expected for C3 plants and animals, demonstrating the need to examine both δ15N and δ13C, in order to separate freshwater fish and forest resources.The fauna from BLD fall broadly into four groups: wild browsers  dating to between 1426 and 1942 years cal. AD  is within the range of values reported for diet–collagen spacing, potentially indicating reliance on these domesticates49. However, δ15N values ranging from 13.4 to 16.9‰, and three individuals with δ15N values higher than 15‰, as well as the riverine setting and modern and historical evidence that the site was a fishing camp  produced results at the limit of statistical significance. This is likely a product of the small sample sizes available. The metabolic routing bias of bone collagen δ13C towards protein components of the diet (at the expense of carbohydrate and fat inputs), and importance of high protein freshwater fish in diets of the measured individuals, means that we can expect that consumption of this low-protein plant resource was actually greater than it appears in the bone collagen values of Fig. 4 plant component, which must have contributed to the diet of Late Iron Age humans and domestic animals in the Inner Congo Basin from at least the 15th century cal. AD onwards.A visible negative correlation between human δood Fig. , althoug51. Tooth enamel was sampled from human second and third molars enabling the investigation of diet during late childhood–early adolescence. Furthermore, given that tooth enamel δ13C reflects the isotopic composition of the whole diet , as opposed to the dominance of the protein signal in bone collagen, tooth enamel δ13C enables additional dietary resolution  are indicative of a dominance of C3 food sources, possibly yams, plantain, or oil palm grown in slightly more open conditions53, or perhaps a mixed diet of closed canopy and freshwater resources. There is no clear evidence for dietary reliance on C4 plants in any of the human tooth enamel samples analysed from IMB, LON, or BLD.Faunal and human δTNW Fig.  reveal d4 consumption somewhat surprising given the collagen data. For four individuals from BLD, it was possible to analyse both bone collagen and tooth enamel to investigate tissue-specific or age-related dietary differences. One individual  demonstrates a predominant reliance on C3 or freshwater food sources from mid-late childhood through to adulthood giving a δ13Cenamel of −11.7 and δ13Ccoll value of −21.0‰. Interestingly, however, BLD 83/1 individual 1 δ13Cenamel value (−13.6‰) is consistent with a reliance on tropical rainforest and freshwater resources, but the accompanying δ13Ccoll value (−17.4‰) reflects a C3/C4-based protein diet. A similar shift is also seen when comparing enamel and collagen results for BLD 83/8  and BLD 83/10 . These distinctions could be a product of either (i) the fact that low-protein rainforest plant resources are more visible in the tooth enamel, while higher-protein animals eating C4 plants  or higher δ13C freshwater resources, with higher δ13C values are more visible in collagen or (ii) greater consumption of C4-based resources in adulthood compared to childhood.As enamel carbonate is reflective of all food sources it would not be affected by a high degree of fish consumption, making this lack of evidence for C13C enamel value of −3.2‰ that clearly indicates that C4 resources made the dominant contribution to the diet of this individual. Unfortunately, it was not possible to analyse bone collagen from this individual, but multiple teeth (M1–M3) were analysed to investigate diet throughout childhood. The results , but cannot specifically be assigned to pearl millet (Pennisetum glaucum), wild finger millet, (Eleusine africana), or domestic finger millet (Eleusine coracana), as they all have in common compound granules and/or markedly polygonal shapes with mean metrics <10 µm  from the MTNW individual and a total of 38 starch granules and 9 phytoliths were retrieved . Another common starch granule type identified is parabolic and/or oblong elongate  come from one tooth (M1) and they are characterised as medium to large, brown globular bodies, with tuberculate to echinate projections. These large, brown phytoliths with variably tuberculated to echinated spines are referenced in the nutshell of Elaeis guineensis  fall into three groups based on their δ13C values: those with δ13C values < −27‰; a single food fragment with an intermediate δ13C value of −24‰; and two food fragments with δ13C values around −9‰ , has a relatively high δ15N value (7.8‰) compared to those of herbivores from the site, perhaps indicating that C4 plant resources like pearl millet and sorghum consumed by humans were growing in different soil conditions compared to the plants eaten by wild and domestic herbivores.The charred food fragments recovered from contexts at BLD ((Pennisetum glaucum) in Iron Age pits in Southern Cameroon18 was one of the most significant findings of the past two decades in Central African archaeology. The discovery sparked debate over the environmental context for early agriculture56, contributing to an increasingly complex narrative for the settlement of the Central Africa rainforest58. Yet direct assessments about the degree to which these early farming communities, particularly in the DRC, relied on C4 resources are limited17. Our data enable direct exploration of the adoption of agriculture at different points during the Iron Age in the DRC and highlight substantial regional variability, particularly with regards to uptake of C4 crops into the diet. The IMB individual, indirectly dated to ~2050 BP  in the dental calculus of this individual indicate that this C4 signal represents the use of sorghum possibly acquired through interactions with agricultural groups in eastern Africa. Nevertheless, the human remains date to 813 ± 35 BP, a time when the region is believed to have been covered by lowland Guineo-Congolain rainforest64. Moreover, the presence of starches from Dioscoreaceae and Asphodelaceae in the dental calculus, as well as the association with wild rainforest fauna64 indicate ongoing contributions of forest resources to the lifeways of this Late Iron Age population. Evidently, the adoption of cereal crops into subsistence economies across tropical Africa was not uniform, displaying regional variation and incorporation into existing, dynamic lifeways.The importance of undertaking a multidisciplinary, contextual approach to the emergence of food production in Central Africa is further indicated by the data from the northeastern DRC site of MTNW. In contrast to the western DRC sites, the stable isotopic data from this individual highlight a clear overall dietary reliance on C4 crops to varying degrees. For the Matangai Turu individual this likely reflects complex forager–farming interactions and exchanges during this time, which have been increasingly recognised in genetic studies66. In contrast, there is presently no evidence for pre-Iron Age indigenous settlement in the Inner Congo Basin, though the local agency of the Late Iron Age populations adapting to the western portion of the region should not be underestimated. Tropical forests in the DRC, as elsewhere67, were home to diverse groups who developed a range of strategies to procure and produce food. We can only begin to look at these dynamic, contextually dependent strategies if we move away from sweeping narratives based on genetic or linguistic data to focus on direct evidence from archaeology, archaeobotany, archaeozoology, and biomolecular methodologies relating to the actual significance of different resources to diets across time and space.The results of this study reveal a diversity of subsistence practices spanning the Central African Iron Age that involved the incorporation of incoming C70 and are increasingly appreciated as essential, but now threatened, carbon ‘sinks’ for the continental and global carbon cycle71. Consequently, there has been substantial discussion about the relative sustainability and antiquity of intensive agricultural land use73 versus mixed agricultural, agroforestry, and hunting strategies in the region75. Despite frequent NGO or government calls to focus on productive cereal monoculture to meet growing populations in West and Central Africa76, it is clear that mixed use of C4 plants, wild resources, rainforest economic plants, and freshwater resources have characterised subsistence practices in the western DRC for over 2000 years. Indeed, the continuation of heterogeneous food production strategies could prove crucial in ensuring long-term food security in tropical Central Africa, as well as the survival of Congolese environments crucial to the global carbon cycle77 pan-African precipitation78, and global biodiversity79.In this way, we can also begin to properly understand the wider significance of ongoing agricultural adaptations in Central Africa. The Congolese portions of the Central African rainforest are considered some of the most vulnerable to climate change13C and δ15N measurements were obtained for human burials from LON (n = 1), BLD (n = 11), and MTNW (n = 1). Totals reflect results taken forward for interpretation. For BLD, two results were excluded from final analysis due to poor preservation (for full details see: “Results” section and Supplementary Table n = 10) to establish a dietary baseline, including domestic browsers (goats), wild browsers (duiker), and fish. To further explore dietary intake, human tooth enamel was sampled from IMB (n = 1), LON (n = 1), BLD (n = 11), and MTNW (n = 1). Due to differential preservation, it was only possible to generate both a bulk collagen and tooth enamel results for four human burials from BLD, and the single individual from LON. In addition, animal tooth enamel from BLD (n = 6) was analysed for δ13C and δ18O to aid the interpretation of human values.Four Iron Age sites were selected for study from across the DRC Fig. : IMB, LO80. Owing to partly waterlogged conditions, the level of organic preservation at BLD is far higher than at sites located above the floodplain, and a number of human burials have been excavated80  were broken into small pieces and adhering soil was removed by abrasion using a sandblaster. Samples were demineralised by immersion in 0.5 M HCl for 1–7 days. Once demineralisation was complete, samples were rinsed three times with ultra-pure H2O. The residue was gelatinised in pH 3 HCl at 70 °C for 48 h, and the soluble collagen solution Ezee-filtered to remove insoluble residues83. Samples were lyophilised in a freeze dryer for 48 h. Where sufficient material was available, ~1.0 mg of the resulting purified collagen was weighed, in duplicate, into tin capsules for analysis.Human and faunal bone collagen was extracted using a modified Longin13C and δ15N ratios of the bone collagen were determined using a Thermo Scientific Flash 2000 Elemental Analyser coupled to a Thermo Delta V Advantage mass spectrometer at the Isotope Laboratory, MPI-SHH, Jena. Isotopic values are reported as the ratio of the heavier isotope to the lighter isotope (13C/12C or 15N/14N) as δ values in parts per mill (‰) relative to international standards, VPDB for δ13C and atmospheric N2 (AIR) for δ15N. Results were calibrated against international standards of  and a laboratory standard . Based on replicate analyses long-term machine error over a year is ±0.2‰ for δ13C and ±0.2‰ for δ15N. Overall measurement precision was studied through the measurement of repeats of fish gelatin . The faunal (n = 8) and human (n = 12) bone collagen results from BLD and LON are presented in Supplementary Table 46.The δ2O and centrifuged before 1 mL 0.1 M acetic acid was added for 10 min. After this, samples were rinsed with ultra-pure H2O, for a total of three washes85. After the final rinse, each tube was placed in a freeze drier for 4 h. In addition, an in-house standard of equid tooth enamel was processed alongside the samples of this study. Approximately 2 mg of the pretreated sample was weighed out into 12 mL borosilicate glass vials for analysis.Approximately 10 mg of tooth enamel powder was obtained from sampled teeth using gentle abrasion with a diamond-tipped drill along the full length of the buccal surface and transferred to a microcentrifuge tube. Teeth were sampled from both humans and fauna (where available) across the four sites. Second and third molars were preferentially sampled from humans providing a long-term insight into juvenile diet, and avoiding the weaning effect potentially visible in first molars. Tooth enamel samples were pretreated with 1 mL 1% NaClO for ~60 min. The samples were rinsed three times with ultra-pure H°C, sample CO2 evolved and was analysed for stable carbon (13C/12C) and oxygen isotopic ratio (18O/16O) composition using a Thermo Gas Bench 2 connected to a Thermo Delta V Advantage Mass Spectrometer. Carbon (δ13C) and oxygen (δ18O) stable isotope values were calibrated against international standards IAEA NBS 18 , IAEA 603 , IAEA CO8 , and USGS44 (δ13C = ~−42.1‰) registered by the International Atomic Energy Agency. Machine error based on the analyses of standards is ±0.1‰ for δ13C and ±0.2‰ for δ18O. Overall measurement precision was assessed through repeat measurements of MERCK CaCO3  and an in-house equid tooth standard .Following reaction with 100% phosphoric acid at 70 54; Fig. 54, and the Ca:P ratio was 2:1–1:7 indicating hydroxyapatite. We present microbotanical materials released from the calcified matrix after thorough decontamination protocols and decalcification in a cleanroom laboratory54, as well as microbotanicals still trapped in the calculus matrix, but visible enough to have their two dimensional morphology identified. Identifications were made according to published morphometric classification criteria for the identification of ancient starch from sub-Saharan plants55.Dental calculus was processed from three mandibular molars M1, M2, and M3) from the MTNW individual. Images of the mineralised plaque prior to removal from the teeth, as well as those from contaminant starch granules and phytoliths are published elsewhere  and IAEA-USGS24 (δ13C –16.05 ± 0.04‰). Measurement uncertainty in δ13C values was monitored using three in-house standards: LEU , GLU , and MIL ) was determined to be ±0.06‰, accuracy or systematic error (u(bias)) was ±0.11‰, and the total analytical uncertainty in δ13C values was estimated to be ±0.13‰, using the equation presented in Supplementary material  and IAEA-N-2 (δ15N 20.3 ± 0.2‰). Measurement uncertainty in δ15N values was monitored using three in-house standards: LEU , GLU , and MIL . u(Rw) was determined to be ±0.18‰, u(bias) was ±0.59‰, and the total analytical uncertainty in δ15N values was estimated to be ±0.61‰.The nitrogen contents of the samples were calculated based on the area under the N86. Radiocarbon ages were calibrated to calendar timescale using OxCal 4 (ref. 87) and IntCal13 atmospheric calibration curve88 , in order to improve understanding of their chronology. Samples were pretreated using previously published methods 4 ref. 8 and IntCr R (version 3.5.3).Sample size was determined by archaeological sample preservation and availability. Data for human bone collagen were analysed using Pearson’s Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Plasmodium falciparum genetic diversity is key to understand evolutionary pressure, measure the impact of elimination campaigns, monitor drug resistance, etc. A common genotyping method is to PCR amplify microsatellite markers or polymorphic genes, such as the msp family, the size of the resulting amplicon(s) being variable between different isolates [The characterization of isolates . Amplicoisolates . AlternaP. falciparum’s most polymorphic gene family: the var genes [var genes have a similar organization consisting in a succession of Duffy binding-like (DBL) and cysteine-rich interdomain region (CIDR) domains with the near ubiquitous presence of DBLα subtype  which is able to infer the posterior probability that each location in an isolate’s DBLα type amino acid sequence is most closely related to every other DBLα type [α found in an isolate to provide an estimate of the expected proportion of relatedness between isolates. These proportions were then aggregated to provide estimates of an isolate’s DBLα repertoire that most closely matched each country, hence attributing a geographical origin to each isolate  was proportionally represented in the Top 100. The overall mind-boggling genetic diversity of DBLα sequences make these specific “conserved” DBLα even more interesting.While most DBLatabases . Althougp B or C , the DBLα could be in linkage disequilibrium with drug resistance alleles [α were associated with drug resistance markers, indicating that other selection forces are at play and remained to be discovered. Interestingly, they identified conserved DBLα on chromosome 6, in which long-range haplotypes have been reported in African P. falciparum genomes [What is driving this selection? Some DBL alleles . However genomes .α types analysis. Their JHMM approach is promising when dealing with relatedness between sets of sequences, even though its computational cost remains to be lowered. Comparing DBLα sequences led to a clustering similar to what was observed using WGS in Africa, for a fraction of the cost. To further investigate the ability of DBLα comparisons to track P. falciparum populations, more samples are needed, especially from Ethiopia and the Democratic Republic of The Congo, where divergent parasites have recently been identified [In summary, Tonkin-Hill and colleagues produced the first global scale DBLentified . Follow-α-based genotyping studies, not just for “varologists” but most epidemiological studies.To conclude, Tonkin-Hill and colleagues provide compelling evidence for further DBL


Here we report that magnetic analysis of the Pook’s Pebbles samples revealed unique electric discharge signatures. Sectioning of Pook’s Pebbles from the Chicxulub ejecta from the Albion Formation at Belize showed that different parts of Pook’s Pebbles had not only contrasting magnetization directions, but also sharply different level of magnetizations. Such behavior is indicative of electric discharge taking place sometimes during the formation of the Chicxulub ejecta blanket. In addition, some of the Pook’s Pebbles’ surface had recrystallized down to 0.2 mm depth. This is evidence of localized extreme pressures and temperatures during the fluidized ejecta formation which was imprinted in the outer layer of Pook’s Pebbles. Recrystallization caused formation of nanophase iron along the surface, which was revealed by mapping of both natural remanent magnetization and of saturation remanence magnetization signatures. While the spheroids’ magnetization orientation is consistent with reversed magnetic field at the time of impact, the study of the Pook’s Pebbles provided, in addition, new evidence of electric charging during the vapor plume cloud processes.Chicxulub impact (66 Ma) event resulted in deposition of spheroids and melt glass, followed by deposition of diamectite and carbonate ejecta represented by large polished striated rounded pebbles and cobbles, henceforth, called Albion Formation The impact produced a globally distributed ejecta layer, and it ranks as the primary cause of this major mass extinction event, which resulted in resetting preexisting ecosystems3. Exposures of K-Pg boundary impact ejecta deposits were found in Belize4. In northern Belize the impact event created a spectacular outcropping of continuous ejecta blanket in the type site named as Albion Formation6. The Albion Formation ejecta blanket contained two lithostratigraphic subunits: The Spheroid Bed, and Diamictite Bed, which overlie the Cretaceous Barton Creek Dolomite. In Northern Belize, there are also present discontinuous ballistic ejecta deposits. The latter are found in abundance southeast of the Chicxulub impact crater center, approximately ~475 km, in the Cayo District of Central Belize  boundary, is linked to a carbonaceous chondrite meteorite collisionize Fig. . These bize Fig. . This di7. The earliest process involves the rapid expansion of a volatile-rich cloud producing a deposit of relatively uniform thickness (Spheroid Bed). The second emplacement process also involves a volatile-rich cloud, but with less velocity and with greater mass density traveling as a debris flow, and a larger range of deposited grain size (Albion Formation Diamictite Bed)1. Beyond a radius of ~360 km (or about four crater radii) the deposition changed into coarse, sometimes well-polished and striated pebbles and cobbles forming the proposed name of Albion Formation Pook’s Pebble Bed, being the type locality the Pook’s Hill Lodge in Teakettle Belize. This locality was first identified in the Cayo District of Central Belize1 and it was proposed that the process that deposited the Pook’s Pebble Bed involved fluidized ejecta with contrasting velocities . The depositional emplacement processes relate to three ejecta transport regimes. The low energy part of the condensing impact vapor plume (mixture of carbonates and water vapor) as it expanded beyond the advancing ejecta curtain entrained small clasts of the Albion Formation Spheroid Bed. The ejecta curtain material was transformed by heat due to interaction with the hot condensing gases and transported as debris flow to form the Albion Formation Diamictite Bed. High-velocity abrasion of large limestone/dolostone fragments, at relatively lower temperature than Diamictite Bed, resulted in deposition of the Pook’s Pebble Bed.The composition and stratigraphy of the ejecta in the southern Yucatan Peninsula suggest that three processes were involved during the ejecta deposition at different crater radii from the center8. The Albion Formation Pook’s Pebble Bed is about 4 m thick, and is a matrix- and clast-supported sub-to well-rounded pebbles and cobbles. More than 50% of these carbonaceous pebbles have distinctive pits ranging from 1 mm to 1–2 cm in size. Portions of these pebbles appear to have smooth polish with defined lineation that grade into striations  that were part of the Albion Formation unit: The lowest unit is 1 to 2 m thick spheroid bed that follows the preexisting karst topography and contains abundant accretionary lapilli. The next unit deposited was 8–15 m thick Diamectite bed material that includes matrix supported clasts of angular to sub rounded shape, some of them with well-preserved striated surfaces. This unit contains clasts over 5 m in size with accretionary rinds Fig. . In centns Figs. –S6. The ace Fig. . The mic11. Dolomite-containing spheroid are likely altered impact-derived accretionary lapilli5 from the same event. The distance of the material collection site is little more than three Chicxulub crater’s radii from the crater center  representing devitrification products from the impact glass that formed during the K-Pg Chicxulub impact crater formationter Fig. , Fig. 3Cter Fig. .Figure 3model 7300, at GSFC/NASA)  Fig. . We anal13 and thus it is likely that they contribute to the magnetization of this carbonaceous material. Oxygen-deficient water moving through the pores dissolved and re-precipitated nano-sized iron oxides around the preexisting iron oxide grains. These pebbles were abundant near the type locality Pook’s Hill Lodge in central Belize  samples have pits, several (35%) have pitting that covers half of the clast14. Pits are elliptical to circular depressions with abrupt rims. The plastically deformed larger pits look like thumb impressions made in soft clay14. Typical pits are 0.3–2.0 cm in diameter and are about half as deep as they are wide. In a few examples the pits are much deeper, forming 0.5–2 cm deep holes that are 0.1–0.5 cm in diameter14. All Pook’s Pebbles have patches of a white, chalky calcite crust that covers on average 24% of the clast. The chalky crust is a few mm thick and composed of mostly 40-µm sized calcite crystals14. The crust also contains about 1% sand and pebble-sized clasts (same lithology as Pook’s Pebbles), which are abundant in a few (8%) samples. Patches of the crust are a gray, translucent, coarse (100 µm-sized) calcite14. A few (8%) examples of this denser crust contain vugs 1–3 mm in diameter and laminations with truncations. Nearly half (43%) of the examined clasts have one facet (and only one) that is mostly covered with crust. Many (42%) of these clasts with crust on one side have extensive pitting on the opposite side. Where the crust is missing, the clast surface is typically polished and/or striated and it is apparent that the crust has been stripped away in the process. A fine polish occurs in patches and rarely covers the entire clasts. In several (~15%) examples, the harder gray translucent crust is also polished, including the small fragments that appear embedded in the crust. Striations are common, occurring in parallel sets and multiple directions, typically within distinct 2–10 mm wide gouges. These gouges also occur in parallel sets, resulting in a striated surface. Many examples of striated gouges have curves and kinks, or end in an abrupt, angular facet.The Albion Formation Pook’s Pebble Bed is material that is composed of sub and well-rounded, polished, pitted, plastically deformed and striated pebbles and cobbles in a clay rich matrix with degraded impact glass green spherules. We analyzed 20 pebbles with polish and/or striations with clast diameters ranging from 4 to 25 cm (mean diameter of 8.6 cm). The Pook’s Pebbles carbonates come both in white and pink colors, some with microfossils. Nearly all (90%) Pook’s Pebbles were fine (~5 µm) grained pink recrystallized limestones with annealed carbonate crust shown by its polish Fig. . The pinWe prepared a thin section from one of the Pook’s Pebble carbonate clasts (verified by reaction with hydrochloric acid). Then used a non-magnetic saw to cut one larger  representative Pook’s Pebble Fig.  into thrWe obtained two cylindrical core samples from Pooks Pebble 04 Fig.  using a 15. The highest magnetization level (0.175–0.252 Am2/kg) at 2 T external magnetic field was obtained from the green devitrified impact glass. Other three components overlapped with each other, however they had clearly lower magnetization than the green devitrified impact glass spherules. The magnetization of limestone/dolomite fragments ranged between −0.052 and −0.017 Am2/kg, while carbonate spheroids had a larger spread – between −0.085 and 0.015 Am2/kg. The pink carbonate Pook’s pebbles Bed spheroids had slightly higher magnetization level – between 0 and 0.051 Am2/kg.We measured saturation magnetization of four main components of the Albion Formation Spheroid Bed: green devitrified glass spherules, limestone fragments, white spheroids, and pink spheroids. This was done in order to check if, magnetically, these ejecta materials differ and the magnetic properties could be used as a unique identifier for ejecta blanket deposits. These measurements show that this technique could be used as a signature for impact ejecta deposits detection by planetary rovers−5 Am2/kg), which did not change significantly after cleaning with the 2 mT alternating field  oriented Pook’s Pebble bed carbonate spheroids were collected from the outcrop in Central Belize and analyzed for direction and intensity of NRM Fig. . Remaneneld Fig. .Figure 4Several geologic thin sections were made from the carbonate pebbles and cobbles from the Albion Formation Pook’s Pebbles Bed. The thin sections were examined under the cross polarized light and contained coarse grained minerals. High order of interreference colors confirmed the prevalence of calcite grains. Interestingly, these crystals contained clear twinning along the surface of the pebble Fig. , down to−9 A m2/kg to its maximum 2.02 10−6 A m2/kg. While the average value was 1.68 10−7 A m2/kg, the median was 7.96 10−8 A m2/kg. Specimens containing the surface material were distinctly more magnetic and varied in magnetization intensity from its minimum value 4.49 10−8 A m2/kg to its maximum 2.02 10−6 A m2/kg. While the average value is 2.90 10−7 A m2/kg, the median was 1.75 10−7 A m2/kg. These parameters are all higher than the cubes from the inside that varied in magnetization intensity from its minimum value 1.58 10−9 A m2/kg to its maximum 2.05 10−7 A m2/kg. While the average value was 5.50 10−8 A m2/kg, the median was 4.38 10−8 A m2/kg.; see also the coloration scale in Fig. The interior slice cut from 12 cm carbonate Pook’s Pebble for magnetic analysis, showed white interior while the surface was distinctly pink Fig. . 52 subs−6 A m2/kg to its maximum 3.57 10−5 A m2/kg. While the average value was 1.36 10−5 A m2/kg, the median was 9.93 10−6 A m2/kg. Specimens containing the interior material were distinctly less magnetic and varied in magnetization intensity from its minimum value 1.62 10−6 A m2/kg to its maximum 4.23 10−6 A m2/kg. While the average value of latter was 2.23 10−6 A m2/kg, the median was 2.02 10−6 A m2/kg. These interior parameters are all lower than the cubes from the outside  are significantly larger than Msr level of the interior (average Msr = (2.23 +/−0.70) 10−6 A m2/kg). See Tables After giving the cubes saturation magnetization inside 2 T magnetic field, the Msr intensity showed similar enhancement Fig. , with di16. In most of the samples that originate in terrestrial conditions with geomagnetic field, such ratio is near 0.0217. Even though the Pook’s Pebble Bed samples most likely did not reach temperatures high enough to acquire strictly TRM for all of the samples, the ratio  relates to the magnitude of the geomagnetic field that magnetized the rock25. Because the geomagnetic field during its history is generated by geodynamo, the magnitude of the flux on the Earth’s surface stays within tens of microtesla. Such flux intensity magnetizes terrestrial rocks during their cooling renders the acquisition of TRM to about 2% of its saturation magnetization, see Kletetschka and Wieczorek (2017)16 and related equations. This value was used as our choice to split the NRM/Msr values in Fig. 26. The non-homogeneous magnetic fields of electric currents could have therefore magnetized portions of the material while other material was demagnetized. Electric current magnetizes preferably low coercivity grains as this happen in low temperature26. Evidence of magnetization by electric current is supported by analyzing the alternating field demagnetization spectra -containing dust reduced to hematite with large magnetization potential. The magnetic characterization of the Albion Formation Pook’s Pebble revealed that the directional and magnitude changes in magnetization of sub-fragments of Pook’s Pebbles is consistent with exposure to electric currents that magnetized/demagnetized portions of the material with a non-homogeneous magnetic field, which may provide insights into the debris cloud environment that this impact material was exposed to. Furthermore, the magnetization signature of the Albion Formation Spheroid Bed, show that this technique could be used as a signature for impact ejecta deposits detection by planetary rovers, and an instrument to be capable to make this type of measurement could enhance the payload for future planetary missions.Given that the Chicxulub Albion Formation Pook’s Pebbles’ surface features are superimposed on the ablation features, we propose that they were formed by particle interactions and collisions as the ejecta passed through a near-surface debris cloud and struck the regolith-covered Cretaceous land surface. Required ejection velocities of 1–2 km/s to reach Belize produce trajectories that that reach altitudes of approximately 100 km. Twinning along the Pook’s Pebbles’ surfaces indicates that some of the them experienced shock pressure up to 500 MPa. We found that devitrified impact glass has distinct paramagnetic enhancement. This property can be utilized as an indicator for future planetary robotic missions, where detection of anomalous paramagnetism can lead to identification of impact glass on the planetary surfacesSupplementary information.


Drosophila dopaminergic neurons (DANs) reinforce memories of unique valence and provide state-dependent motivational control [Different types of  control . Prior s control , 3. Here control  to recon control , 6. Trac •Nanoscale anatomy reveals additional subtypes of rewarding dopaminergic neurons•Connectomics reveals input specificity to subtypes of dopaminergic neurons•Axon morphology implies dopaminergic neurons provide subcompartment-level function•Unique dopaminergic subtypes serve aversive memory extinction and sugar learning Otto et al. use electron microscope resolution connectomics to describe the structure and organization of neurons providing synaptic input to functionally discrete subtypes of dopaminergic neurons. The nanoscale anatomy reveals further anatomical and functional specialization of dopaminergic neurons, which is confirmed with behavioral experiments. Drosophila, anatomically discrete dopaminergic neurons (DANs) innervate adjacent compartments of the mushroom body (MB) [In adult ody (MB) . In someody (MB) , 10, 11.ody (MB) , 13, 14.ody (MB) , 15, 16.We used a recent EM dataset of a full adult fly brain (FAFB)  to identPart 1: 3D representations of all canonical RHS DAN skeletons traced to identification in this project within the MB neuropil. PPL1-γ1pedc DAN , PAM-γ5 , PAM-β′2a . Part 2: 3D representations of skeletons of newly identified subtypes of PAM DANs. Axons from the different subtypes innervate distinct areas in the γ5 and β′a compartments and their dendrites occupy unique areas in the SMP. Note: for visibility the LHS axonal projections are omitted and only the proximal commissural axons are retained.We noticed when reconstructing PAM-γ5 DANs that their dendrites occupied different areas of the superior medial protocerebrum (SMP) , that thThe four reviewed PAM-β′2a DANs could also be clustered into three groups with commissure crossed, overall morphology, and region of compartment innervation again being the distinguishing features . TracingReconstructing the individual right hand PPL1-γ1pedc DAN revealed that its dendrites occupy locations distinct from those of PAM-γ5 and PAM-β′2a DANs K. This s3D representation of the traced RHS PPL1-γ1pedc DAN skeleton within the MB neuropil. The dendritic arbors are labeled by the different areas of neuropil in which they receive synaptic input: SIP (blue), SMP (violet), dorsal CRE (green), ventral CRE (red). The ipsilateral axon, the soma and soma tract are gray. Video V6 details locations of input from MBONs.We next traced 821 neurons providing input to postsynapses identified in the dendrites of the PAM-γ5, PAM-β′2a, and PPL1-γ1pedc DANs A. Since Reassuringly, input selectivity of PAM-γ5, PAM-β′2a, and PPL1-γ1pedc DANs largely reflected the relative overlap of their dendritic fields. All the DANs receive unique inputs A–S2D. HoOur sampling suggests that despite there being 20 PAM-γ5 DANs and one PPL1-γ1ped DAN, there are only approximately twice as many inputs to all PAM-γ5 DANs compared to the PPL1-γ1pedc DAN B. HoweveThe observed connectivity could be confounded by the incompleteness of reconstruction. However, plotting the synapses identified following standard and extensive review suggests that each iteration of review adds synapses that are evenly distributed across a DAN’s dendritic arbor H. NevertWe next clustered the input neurons based on their morphology using a three-step approach . Using sPart 1: 3D representations of the skeletons of 821 neurons that provide input to PPL1-γ1pedc, PAM-γ5 and PAM-β′2a DANs. Neurons are shown within the whole brain neuropil color coded according to the first step of coarse clustering . Note: SEZONs are shades of blue, LH-related neurons are shades of yellow, and OTHERS confined to the superior part of the brain are in shades of violet. Part 2: 3D representation of the skeletons of the fine clusters of input neurons that are studied in detail. Colors correspond to those in Figure 3: LHON01, LHON02, and LHON-AD1b2 neurons are shown in shades of yellow, MBON-γ4γ5 s in green, the RHS MBON-γ5β′2a is coral, SEZONs are shades of blue, and OTHERS are shades of violet.Annotating the DAN clustering with input neuron identity revealed that the γ5β′2a MBONs specifically provide feedback (fb) input from the MB to our previously defined PAM-γ5(dd) subtype K. We theToward assigning functional relevance to the identified input pathways, we used NBLAST  to screePairing odor exposure with optogenetic activation of PPL1-γ1pedc or PAM-γ5, PAM-β′2a DANs can produce either aversive or appetitive odor memories, respectively . We therThe γ5β′2a and γ4γ5 MBONs both have a dendrite in the γ5 compartment , 11. In Tracing of DAN inputs also identified selective connectivity with a few other MBONs, some of which were unknown C. MBON-αSynthetic activation of the SEZON lines also produced different learning phenotypes. Activating SEZON01 neurons formed aversive memory and these connect to PPL1-γ1pedc and some PAM-γ5, but not to γ5 DANs reinforcing sugar memory (see below). Stimulating SEZON03 neurons formed appetitive memory and these SEZONs synapse onto PAM-γ5, but not PPL1-γ1pedc. SEZON02 neuron activation did not implant significant memory of either valence and appears to connect weakly to all three classes of traced DANs K.Shibirets1 transgene [Despite their specificity, we expect some of our identified GAL4 drivers to express in our traced neurons of interest, and additional similar neurons in a fascicle. For example, a SEZON line could label a collection of ascending neurons representing both tasteful and distasteful gustatory stimuli . Labelinransgene  to blockransgene , and MB3ransgene . Blockinv and CREd branches. Since the CRE branches are closest to the primary axon, input from other MB compartments may be particularly salient to PPL1-γ1pedc , SMP (purple), dorsal CRE (green), ventral CRE (red). The ipsilateral axon, soma and soma tract are gray. The dendritic location of input synapses from MBONs are indicated, with most targeting the CRE portion of the dendritic field. MBON-γ4y5 , MBON-γ2 , MBON γ3β′1 , MBON γ3 , MBON γ2α′1 , MBON-α3 .Based on prior findings, we hypothesized that PAM-γ5(fb) DANs, which receive recurrent feedback from MBON-γ5β′2a, would be required for memory revaluation  and otheShits1 disrupted aversive memory extinction and/or sugar learning .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Scott Waddell  brain (FAFB) [https://catmaid.readthedocs.io/en/stable/) [Neurons were reconstructed from a serial section transmission electron microscopy (ssTEM) volume of a full adult fly (n (FAFB)  using CAstable/) , 42. Constable/) , 37, 49,stable/) , where fDrosophila, presynapses have been found on fine axonal processes [Synaptic sites were identified based on three, previously described criteria  and revirocesses , boutonsrocesses , and othrocesses . It has rocesses . The proDrosophila brain. DAN dendrites are very thin and have a dark/granular texture, which increases the likelihood of missing branches and synapses. We therefore scrutinized completion and postsynapse annotation for 7 PAM-γ5 DANs , 2 PAM-β′2a DANs and the PPL1-γ1pedc DAN. Following this extended reconstruction and revision effort, we are confident that we have annotated all identifiable postsynapses on these selected DANs. Comparing data obtained from the regular review protocol to that from our extended review effort showed that regular review captured ∼30% of the postsynapses on more than 60% of all cable. We also analyzed the placement of old (regular review) and new (added following extensive review) synapses, by measuring their geodesic  distance to the dendritic root  β′2a DANs, and γ5 DANs. Although the number of γ5 DANs has been estimated to be between 8 and 21 [tic root H. This aWhen a postsynapse was annotated on a DAN, a single- node profile was placed in the center of the presynaptic cell, unless a neuron or fragment was already present. To reconstruct upstream neurons from these seminal nodes we randomized the sampling order from each postsynapse within the total population on a neuron-by-neuron basis. For the reviewed PAM DANs (18 γ5 and 4 β′2a) we typically traced over 85% of the input neurons to identification from annotated postsynapses on a DAN arbor  or natverse [http://jefferis.github.io/rcatmaid/). Neuron data from CATMAID were imported using an existing CATMAID plugin for Blender .3D representations of traced skeletons that were obtained as swc files from CATMAID after reconstruction were either plotted using the natverse R tool box, or 3D representations were created and rendered with blender v2.79. Video footage of skeletons was rendered to obtain 3D representations with blender v2.79 and cut with Adobe Premiere Pro2020.https://v2.virtualflybrain.org). NBLAST similarity matrices were calculated comparing both data types and top hits were visualized and manually cross compared. In case of new split-GAL4 SS lines created by Masayoshi Ito (unpublished data), imaging stacks were newly registered with bridging and mirror registrations from the natverse package [EM skeletons were matched to published library GAL4 lines using registered Janelia FlyLight micrograph data from VirtualFlyBrain  and RCatmaid  were used to interface with CATMAID servers and perform morphological analyses. Neuron analyses were performed with natverse functionality (https://github.com/natverse/nat) [https://github.com/jefferis/dendroextras). Tanglegrams were generated using dendextend  [https://github.com/vegandevs/vegan).All analyses were performed in R and Python using open-source software. PyMaid (rse/nat)  or custodextend) . Mantel To analyze and draw conclusions from differences and similarities in large amounts of connectivity or morphology data, the information is represented in the form of distance matrices between each data point in space. Euclidean distance is the direct (bee-line) distance between two points in a Cartesian coordinate system. Manhattan distance between two data points in a Cartesian system is the sum of distances between the coordinates.Ward’s method was used for agglomerative hierarchical clustering (part of R base package). Each datapoint starts in its own cluster and pairs of clusters are merged, moving up the hierarchy. At each step the pair of clusters with minimum within-cluster variance are merged. Connectivity data, as well as morphology data was clustered using Ward’s criterion to compare to clusters formed using average linkage criterion.Average linkage  is another criterion for agglomerative hierarchical clustering. With average linkage clustering pairwise dissimilarities between each element in cluster 1 and 2 are computed and the average of these dissimilarities are considered as the distance between the two clusters. Clusters separated by the smallest distance are merged during clustering, moving up the hierarchy. Both morphology and connectivity data were clustered using average linkage to compare to data clustered with Ward’s criterion.To compensate for different levels of completeness of tracing, DANs were simplified to their longest tree with 200 branch points (the minimum number of branch points throughout the PAM DAN population). Morphological similarity matrices were calculated using NBLAST . HierarcConnectivity information was retrieved from CATMAID after synapse annotation and upstream tracing of input neurons. Only neurons upstream of the dendritic region of DANs with > 50 sampled profiles were included in the analyses. Before clustering the number of synapses annotated on each DAN was normalized to reduce bias in clustering that could arise from the varying levels of tracing completeness and/or natural differences in the number of inputs to the different DANs. Hierarchical clustering was primarily performed using the Manhattan distance between upstream connectivity profiles of DANs with Ward’s clustering criterion. Connectivity data was also clustered using the average linkage criterion for comparison.https://www.scipy.org/ [For PPL1-γ1pedc the x, y and z coordinates of postsynapses on the dendrite were clustered using Ward’s hierarchical clustering in the SciPy package (ipy.org/ ).Silhouette is a graphical representation of the quality of clustering across a range of potential values for k (the number of clusters). Where possible Silhouette was used to select an appropriate number of clusters (it was less useful for morphology clustering). This method measures how similar observations are to their own cluster and how dissimilar to other clusters. The average silhouette width ranges from 0 and 1, with 1 indicating observations are well clustered. To validate DAN clustering the average silhouette width was calculated using the nbclust R package , 53.Morphology clustering of upstream neurons was performed using hierarchical clustering with average linkage criterion. This involved a multi-step approach to account for varying levels of tracing and for the morphological diversity of 821 neurons . Coarse Tanglegrams were generated to visually compare clustering dendrograms produced by different criteria  or clustering based on morphology versus those produced using connectivity. Dendrogram layouts were determined to minimize edge crossing  using dendextend .7 times and each event tested for correlation with the observed data. The number of events where the correlation is higher than between the two original datasets was divided by the amount of comparisons (107) to create a p value. When p values were lower than the significance level, it was concluded that the null model of independence between the two feature spaces could be rejected  that a downsampled DAN clustered with the same group that it clustered with in the full 100% connectivity dataset. These values were then plotted as a stacked bar plot.The Fowlkes-Mallows Index measures the similarity of the content between two different clusterings. The performance of the first clustering is compared to that of the second clustering (which is assumed to be perfect). Exact matches/good performance result in an FMI = 1 .To identify SEZONs and LH-associated neurons, the cable length within the respective neuropil mesh (3D bounding box) was calculated. A cut off of > 60 nodes within the defined neuropil region was required for classification.https://graphviz.gitlab.io/ [https://github.com/markuspleijzier/AdultEM/tree/master/Dendrogram_code) using the Graphviz library with Python bindings provided by NetworkX, (https://networkx.github.io/ [Dendrogram representations of neurons were created as in . Dendrogtlab.io/ ) attemptthub.io/ ).Euclidean distances between MBON-γ5β′2a postsynapses and the closest DAN presynapse were measured and marked with the identity of the morphological DAN clusters . The EucEdge weight distributions describe how many upstream neurons contribute a given number of presynapses to a connection with a postsynaptic neuron (frequency versus number of synapses). Normalizing by the total number of postsynapses details the percentage of the total postsynaptic budget a given number of synapses represents. For example, if a neuron makes 10 presynapses onto a postsynaptic neuron, which has a total of 100 postsynapses, then that upstream neuron contributes 10% of the postsynaptic budget.Identified MBONs were collapsed by type. The number of synapses between DANs and MBONs was normalized by the number of all DAN-MBON connections of the given DAN. Connectivity matrices can be calculated for single branches of a neuron after defining the relevant branchpoints in CATMAID. For the PPL1-γ1pedc DAN, we manually split the dendrite into 4 postsynaptic clusters, as defined from cluster analyses, and recorded the specific connectivity to each of these clusters/branches.A connectivity similarity score between 2 DANs was defined as one minus half of the Manhattan distance between their normalized connectivity patterns . Then the Manhattan distance between upstream connectivity profiles of DANs in the observed data and those in simulated random matrices, both normalized by DAN postsynaptic budget were calculated and means of these distances were compared to obtain a p value describing the similarity of these means. A p value lower than the significance level concluded that the null model of randomized connectivity could be rejected (see A DAN input connectivity matrix was first randomized 10cted see .−3 dilutions of 3-octanol (OCT) and 4-methylcyclohexanol (MCH) in mineral oil. For extinction experiments odor concentrations of 10−6 were used to avoid pre-exposure effects [The odors used for US substitution, sucrose learning and DEET learning experiments were 10 effects . ExperimIn both the behavioral screen and follow-up experiments, neurons were artificially activated to substitute for an unconditioned stimulus in the training chamber of a T-maze. Prior to the experiments, 80-120 1-5 day old mixed sex flies were housed on standard food supplemented with 1% all-trans-Retinal for 3 days before a 20 – 28 h starvation period in vials containing 2 mL 1% agar as a water source and a 2x4 cm strip of Whatman filter paper. During training, groups of flies were exposed to the CS- for 2 min followed by 30 s rest with fresh air, then 2 min of CS+ odor with optogenetic activation of the genetically encoded Channel Rhodopsin with red light exposure. Three red (620-630nm) LEDs  with 3 W maximum power were mounted on the training arm of a T-maze and 1ms pulses were driven at 1.2V with a stimulation frequency of 500Hz, which is flicker free red-light that flies cannot see. For screening, immediate memory testing followed. Flies were transferred back into their starvation vials after training before testing 30 min memory.Prior to the experiments, 80-120 3-8 day old mixed sex flies were starved for 20-28 h in vials containing 2 mL 1% agar as a water source and a 2x4 cm strip of Whatman filter. Flies were transferred to 32°C 30 min before training. During training, groups of flies were exposed to the CS- odor with dry paper for 2 min followed by 30 s of fresh air, then 2 min of CS+ odor exposure with dry sugar paper. Flies were either tested immediately after training or were transferred back into 25°C starvation vials after training prior to testing 30 min memory.Flies were aversively trained with DEET as previously described . In brieExtinction memory was tested as described . In brieTo test memory performance flies were loaded into the T-maze and transported to the choice point where they were given two min to choose between the CS+ and CS- odors in the dark. A Performance Index was calculated as the number of flies in the CS+ arm minus the number in the CS- arm, divided by the total number of flies . MCH and


Invading breast cancer cells transform the mechanics of their surroundings across a panorama of time scales. Living tissues embody a unique class of hybrid materials in which active and thermal forces are inextricably linked. Mechanical characterization of tissues demands descriptors that respect this hybrid nature. In this work, we develop a microrheology-based force spectrum analysis (FSA) technique to dissect the active and passive fluctuations of the extracellular matrix (ECM) in three-dimensional (3D) cell culture models. In two different stromal models and a 3D breast cancer spheroid model, our FSA reveals emergent hybrid dynamics that involve both high-frequency stress stiffening and low-frequency fluidization of the ECM. We show that this is a general consequence of nonlinear coupling between active forces and the frequency-dependent viscoelasticity of stress-stiffening networks. In 3D breast cancer spheroids, this dual active stiffening and fluidization is tightly connected with invasion. Our results suggest a mechanism whereby breast cancer cells reconcile the seemingly contradictory requirements for both tension and malleability in the ECM during invasion. In living tissues, the mechanical coupling between active cells and their passive extracellular matrix (ECM) forges a unique class of hybrid materials. Active and passive mechanical elements are inseparably linked into a dynamic whole that is greater than the sum of its parts. This reciprocity between active forces and the ECM shapes myriad phenomena relevant to human development, aging, and disease (−6 to 10−3 s) (−3 s) (0 to 102 s) , which interrogates the broadband viscoelasticity of soft materials   .α revealed the coexistence of suppressed motion on short time scales and more directed motion on long time scales  on sufficiently long time scales. Superdiffusive motion is not expected from purely thermal fluctuations, where 0 < α < 1, with the lower and upper limits corresponding to purely elastic and purely viscous materials, respectively , which describes the response of the material to small perturbations superimposed onto the prestress (f(τ)f(0)〉 [and corresponding Fourier transform 〈∣f(ω)∣2 ∣〉]. For small superimposed force fluctuations, such a particle will undergo motion whose Fourier-transformed MSD 〈Δr2(ω)〉 is given in T∣2〉 and active 〈∣f(ω)A∣2〉 contributions, i.e., 〈∣f(ω)∣2〉 = 〈∣f(ω)T∣2〉 + 〈∣f(ω)A∣2〉 A∣2〉 . Thus, if(ω)A∣2〉 .G*(ω). To estimate G* on day 6, we assume that F-actin depolymerization completely abolishes active forces, producing motion governed by the generalized Stokes-Einstein relation (GSER) , this assumption is supported by our macrorheology measurements of the frequency-dependent differential modulus in the presence of an imposed prestress (fig. S7), which revealed that increasing prestress increased G* by a frequency-independent, prestress-dependent multiplicative factor.Our FSA requires an estimate of the frequency-dependent shear modulus This analysis revealed substantial stiffening of the ECM through a combination of transient (reversible) and viscoplastic (irreversible) mechanisms. Over the first 6 days in culture, the fibroblasts globally stiffened the matrix by a frequency-independent factor of about 5 , therebyf(ω)A∣2〉 ∼ ω−2. This ω−2 scaling is the anticipated high-frequency scaling for a matrix in which fluctuating internal stresses are generated by contractile motor forces that instantaneously release tension after a Poisson distribution of lifetimes A∣2〉 adopted a weaker frequency dependence, suggesting that the active forces fluctuated with a broad distribution of correlation times ranging from at least 10 to 102 s.Active forces not only alter ECM viscoelasticity but also compete with thermal fluctuations in driving particle motion. In our measurements, active fluctuations played a dominant role at long time scales and were eclipsed by thermal forces on short time scales . In the f(ω)A∣2〉 ∼ ω−2 will prevail at short time scales but will be dominated by the broad frequency dependence of the thermal fluctuations of biopolymer matrices on sufficiently short time scales. Together, these competing effects will generally lead to hybrid mechanics in which active forces drive fluidization of the matrix at long time scales where their fluctuations overshadow thermal forces and stiffen the matrix on short times by bearing steady tension on the ECM  in col/rBM (fig. S9), which reinforces the general principles underlying this phenomenon. Whenever slowly fluctuating processes control active forces, a high-frequency scaling for active force fluctuations 〈∣ the ECM .r2 , with the differential shear modulus taken as the locally linear response function.Because we do not measure the differential shear modulus of the prestressed ECM directly, our calculation of This assumption is not guaranteed but is consistent with optical tweezers microrheology experiments on active biological materials and internally consistent with our results. Studies comparing active and passive microrheology of motor-activated cytoskeleton networks and living cells have demonstrated that the color of the force fluctuation spectrum at thermally dominated high frequencies is consistent with the FDT, despite the fact that the gel exists in a nonequilibrium, stress-stiffened state , we treated the cells with or without TGFβ. We allowed 3D morphogenesis to proceed for 6 days before performing an additional DLSμR measurement and fixing cultures for immunofluorescence confocal microscopy.After 6 days of culture, matrix composition commandingly regulated the outcome of TGFβ-mediated EMT. In both matrices, TGFβ treatment promoted classical molecular signatures of EMT, including substantial up-regulation of vimentin , cadherin expression switching from E-cadherin to N-cadherin , up-regulation of the EMT “master” transcription factors SNAIL and Zeb1 (fig. S10 and table S1), and up-regulated transcription of matrix metalloproteinases (MMPs) (fig. S11). However, despite similar molecular changes in these EMT markers, TGFβ drove invasion only in col/rBM. In col/rBM, treatment with TGFβ promoted collective invasion of aggregates into the matrix, resulting in highly connected 3D networks formed by interacting spheroids . In rBM,Similar to contractile stromal cells, the experimental conditions that drove invasion also promoted time scale–dependent, hybrid matrix remodeling, as observed by the particle MSD measured by DLSμR . In rBM,These hybrid dynamics driven by invasive spheroids accompanied notable changes in the active force fluctuation spectrum and physical organization of the ECM. FSA revealed that in col/rBM, invasive spheroids exerted active force fluctuations over an order of magnitude greater than noninvasive spheroids . ConfocaG* across all frequencies in the thermal frequency regime –mediated cytoskeletal dynamics . Our findings suggest that matrix degradation facilitates long time scale fibril reorganization, which enables active forces to viscoplastically remodel the network to reconfigure transient network cross-links, thereby stiffening the matrix on time scales shorter than fibril relaxation times.To investigate potential confounding effects of these drug inhibitors on proliferation, we performed genomic DNA quantification (fig. S15) and microscopy-based cluster size analysis (fig. S16). ROCK and Rac inhibition had no impact on proliferation (fig. S15). However, MMP inhibition substantially reduced proliferation compared to all other conditions and correspondingly produced the smallest average spheroid size (fig. S16). To explore the potential role of reduced proliferation and smaller spheroid size in the MMP-inhibited condition, we preaggregated spheroids in microwell plates (50 cells per spheroid) and encapsulated the spheroids in col/rBM at a cell density comparable to the final time point of the most proliferative condition (col/rBM without TGFβ). Using DLSμR to probe the matrix dynamics, we found that preaggregated spheroids treated with TGFβ and the MMP inhibitor still failed to stiffen and fluidize the matrix after 6 days (fig. S17). These results suggest that the effect of MMP inhibition on matrix remodeling cannot be accounted for by its effect on proliferation and cluster size.Last, we found that the same molecular mechanisms responsible for matrix fluidization and stiffening were also required for invasion. To quantify collective invasion, we performed confocal microscopy on day 6 and quantified the 3D connected structures formed . Unlike Our results demonstrate an unexpected role for MMPs in regulating matrix stiffness. Matrix stiffening is often viewed as the outcome of an imbalance between the opposing actions of matrix building and matrix degradation ]. We conducted CONTIN and second-order cumulant particle size analysis on these data using the known viscosity of water. The scattering correlation function was consistent with single scattering from a low-polydispersity suspension of particles with mean size close to that reported by the manufacturer.5 cells/ml for all experiments) in ice-cold ECM precursor solution containing 0.25% (w/v) 2.0-μm PEGylated polystyrene beads  and sonicating with a probe sonicator until the gel was completed disrupted. Five microliters of lysis solution was diluted with 20 μl of TE buffer [10 mM tris-HCl and 1 mM EDTA (pH 8.0)] and loaded into a 384-well black-bottom plate. PicoGreen dye  was diluted 200-fold with TE buffer, and 30 μl of diluted reagent was added to each well. The sample plate was mixed by shaking and incubated in the dark for 10 min before collecting spectrophotometry measurements. Fluorescence was read using a well-plate spectrophotometer by excitation with 488-nm light and collecting emission at 520 nm. A standard curve to identify the linear regime was obtained by directly lysing 10 μl of cell suspensions containing defined cell concentrations . Any samples that were outside of the linear regime identified by the standard curve were diluted twofold, and the absolute fluorescence was corrected accordingly.Relative proliferation of MCF10AT cells in 3D ECMs was performed by fluorometric quantification of total genomic DNA using PicoGreen dye. Each biological replicate  dissolved freshly in DMEM/F12 at 10 mg/ml. Col/rBM hydrogels containing 2.0 μm of polystyrene beads were prepared as described in the “Dynamic light scattering microrheology” section above. After pH neutralization, hydrogels (40 μl) were cast in disposable cuvettes and incubated for 30 min at 37°C. After gelation, 300 μl of collagenase-containing media (10 mg/ml) was added on top of the gel. Immediately after addition of the collagenase, DLSμR measurements were performed continuously for 90 min at 37°C, with each time point representing an average of the correlation function over a 10-min interval. After 90 min, a scattering intensity measurement was performed over 24 measurement positions (10 s each) to correct for broken ergodicity in all time points. Time courses were performed on three full reproductions of the experiment.4 cells/ml was added. The plate was centrifuged twice at 200g for 5 min each. Plates were then incubated at 37°C for 16 hours to allow aggregates to form. After 16 hours of aggregation, spheroid formation was confirmed by phase contrast microscopy. Spheroids were removed from the microwells by pipetting up and down with 1 ml of media. This was repeated with a fresh 1 ml of media until nearly all spheroids were removed. Spheroids were collected by centrifugation at 200g for 3 min and then resuspended in col/rBM containing 2.0 μm of beads at a density of 1.2 × 105 spheroids/ml . Forty microliters of each hydrogel solution was cast into disposable cuvettes, and DLSμR was performed on day 0, day 6, and after 24 hours of latrunculin A treatment beginning on day 6, as described for other conditions. The MMP inhibitor GM6001  and TGFβ  were included throughout the duration of culture. For confocal microscopy, spheroid-populated hydrogels were cast in 4-mm-diameter circular silicone molds. On day 6, cells were fixed, stained using TRITC-phalloidin to visualize F-actin, and imaged by confocal microscopy using a 40× oil immersion objective, as described in confocal microscopy and immunofluorescence.To test whether preaggregation of spheroids can rescue matrix remodeling in MMP-inhibited spheroids, aggregates were preformed in low-adhesion 400-μm microwell plates (STEMCELL Technologies AggreWell 400 24-well plates) with 50 cells per spheroid. To prevent adhesion to the plate, microwell plates were incubated with 0.5% BSA in PBS at 37°C for at least 6 hours. The BSA solution was completely aspirated from all microwells. A single-cell suspension of MCF10AT cells in complete MCF10AT media. To each well of the 24-well plate (containing 1200 microwells per well), 1 ml of a suspension containing 6 × 10n) represent distinct cell/gel suspensions cast in either separate cuvettes (for DLSμR) or separate circular molds . For most DLSμR experiments, replicates were collected across multiple cell passages collected on separate days . In some cases, all replicate gels were prepared from the same cell passage. The number of distinct cell passages from which replicates were pooled for DLSμR experiments are as follows : HMFs, 3; MCF10AT +TGFβ in col/rBM, 2; MCF10AT −TGFβ in col/rBM, 2; MCF10AT +MMPi, 2; MCF10AT +Rocki +Raci, 2; MCF10AT +ROCKi, 3; MCF10AT +Raci, 1; and MCF10AT −TGFβ in rBM, 1. For confocal microscopy–based quantification of morphology, all conditions and replicates were prepared from the same cell passage. Qualitative observations of invasive morphology were confirmed for all conditions in at least three separate passages.Replicates . In cases where equal variances were not supported by the data, unpooled variances were used, as indicated. Otherwise, pooled variances were used.Data are represented as either the arithmetic mean or geometric mean (where indicated), with shading or error bars indicating 68% confidence intervals of the mean. Confidence intervals were computed by bootstrapping with replacement using custom scripts written in Python. With one exception (described below), statistical significance was evaluated using a one-sided t test was not used since this does not represent simply a difference of normally distributed variables (it is a difference of differences). Instead, a two-sided parametric bootstrap t hypothesis test was performed using a custom analysis script written in Python. Briefly, for a given gene, the studentized bootstrap distribution of log fold changes in expression (with TGFβ versus without TGFβ) was computed for each ECM by parametrically sampling from the student t distribution (105 bootstrap samples). The empirical distribution of differences in log fold changes (induced by TGFβ) between ECMs was obtained by forming pairwise differences between the two bootstrap distributions. Confidence intervals, CI, representing different significance levels were formed (P = 1 − CI), and the largest confidence interval not encompassing zero was taken as the significance level. Multiple comparisons were then corrected for using the Benjamini and Hochberg false discovery rate correction in R.To evaluate the difference in fold induction of gene expression upon TGFβ treatment between ECMs,


No differences in mortality were observed within the two TTM strategies. These results suggest that an inappropriate translation of TTM protocols from large well-conducted randomized trials into clinical management may result in unexpected effects on patients’ outcome. As for all newly commercialized drugs, epidemiological studies and surveillance programs with an adequate follow-up on large databases are necessary to understand how RCTs are implemented into medical practice.There is a persistent debate on the optimal target temperature to use during cooling procedures in cardiac arrest survivors. A large randomized clinical trial (RCT) including more than 900 patients showed that targeted temperature management (TTM) at 33 °C had similar mortality and unfavorable neurological outcome (UO) rates as TTM at 36 °C in out-of-hospital cardiac arrest patients with any initial rhythm. Since then, several observational studies have been published on the effects of changes in target temperature  on patients’ outcome. We performed a systematic literature search from 1 January 2014 to 4 December 2020 and identified ten retrospective studies , including 5509 patients, that evaluated TTM at 33 °C vs. TTM at 36 °C on the occurrence of UO (n = eight studies) and mortality (n = ten studies). TTM at 33 °C was associated with a lower risk of UO when studies assessing neurological outcome with the Cerebral Performance Categories were analyzed (OR 0.80 [95% CIs 0.72–0.98];  The most severe complication of sudden cardiac death is hypoxic-ischemic brain injury (HIBI) ; most paThe only therapeutic intervention that has shown some benefits to mitigate the sequelae of HIBI is the use of targeted temperature management (TTM); at this moment, three randomized clinical trials (RCTs) have demonstrated a significant improvement in the proportion of patients with favorable neurological outcome when cooling at 33 °C for 24 h was compared to no temperature control or normothermia ,6,7. NevIn clinical practice, one of the most controversial issues remains the target temperature to use during the cooling phase; in 2013, a large RCT including more than 900 patients compared TTM at 33 °C to 36 °C in OHCA patients with any initial rhythm and reported similar mortality and favorable neurological outcome rate in the two groups . If, on We adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis-Protocols (PRISMA-P) guidelines .A systematic literature search from 1 January 2014 to 4 December 2020 was performed on PubMed using the following terms: (“targeted temperature management” OR “TTM” OR “hypothermia” OR “cooling” OR “therapeutic hypothermia” OR “33 C” OR “36 C”) AND . All observational studies published in English in peer-reviewed journals and comparing the use of TTM at 33 vs. 36 °C in adults (>18 years of age) were eligible for inclusion.The research question was: (1) In cardiac arrest survivors (participants), is TTM at 33 °C (intervention), when compared to TTM at 36 °C (control), associated with similar neurological outcome and mortality (outcome) in observational studies (study) published after the “TTM trial” ? We alsopost hoc analyses from the “TTM trial” [Two authors (AM and FA) independently screened study titles and abstracts for potential eligibility and assessed their validity. Disagreement between authors was assessed and resolved through a third reviewer (FST), who reviewed the original text of the article. None of the authors of the original studies was contacted to obtain further information, which were not available in the published manuscript. All M trial”  were excM trial” . EditoriThe level of evidence (LOE) of each study was assessed according to the Grading of Recommendations, Assessment, Development and Evaluations (GRADE) evidence system . The risThe primary outcome of the meta-analysis was the occurrence of unfavorable neurological outcome (UO), whenever it was recorded. UO was defined according to the definition of each study; however, as UO is generally identified as a Cerebral Performance Category (CPC) of 3–5 in most of studies conducted in the cardiac arrest field, a subgroup analysis including only studies reporting CPC 3–5 as. UO was performed. Secondary outcome was mortality, whenever it was recorded; in case of several time-points of mortality assessment, the longest delay from arrest was considered. Subgroups analyses were performed for: (a) Different timing of outcome assessment ; (b) only OHCA patients; and (c) only patients presenting with an initial shockable rhythm.p < 0.05. The I2 was calculated by χ2 test to assess variability due to heterogeneity rather than chance. A substantial heterogeneity was assumed with I2 > 50%. No funnel plot was performed as the total number of selected studies was less than 10.Statistical analysis was conducted by Review Manager 5.3 software. Means of UO and mortality probabilities were obtained by weighting each study by the inverse of variance. The Mantel–Haenszel method was chosen as the reference method for fixed effects analysis. A Z test was carried out to assess the significance of the risk differences. Odds ratio (OR) and 95% confidence intervals (CIs) for UO and mortality were calculated with the Wilson method and placed in forest plots and statistical significance was assumed for A total of 2681 records were identified after the initial search. After the first screening procedure, 48 studies were assessed for eligibility. Of those, 39 were excluded and ten retrospective studies ,23,24,25The characteristics of the selected studies are summarized in Unfavorable neurological outcome (n = 3155) was defined in six studies as Cerebral Performance Category (CPC) of 3–5 ,23,24,25p = 0.04—p = 0.03) than those treated at 36 °C (2 of 0%).Overall, patients treated with TTM at 33 °C showed similar UO than those treated at 36 °C  than those treated at 36°C , ICU discharge (n = 4), at 30 days (n = 2) or six months (n = 2) after arrest . Overall at 36°C .Subgroup analysis analyzing survival at different time-points also showed no differences between the two groups; however, high heterogeneity was observed in some of the analyses. No differences in mortality were observed when mortality was analyzed only in patients with an initial shockable rhythm .post hoc analyses from the TTM database showed any clear advantage of TTM at 36 °C on TTM at 33 °C, including the occurrence of infections, acute kidney injury or seizures [Many centers have adopted a target temperature of 36 °C for the management of cardiac arrest patients suffering from HIBI ,27; overseizures ,30,31. Tseizures ,33,34, aseizures , some stseizures ,28, whicBecause of this discrepancy between a pragmatic RCT and “real life “data, we performed this systematic research and meta-analysis, aiming to summarize how the translation of the TTM-study results in clinical practice may have affected patients’ outcome. Accordingly, we observed a similar mortality rate between the two TTM strategies, but a lower probability of UO when TTM at 33 °C was applied. We based this statement on the analysis of UO as CPC 3–5, because one study reported neurological function using the ability of the patient to follow commands, which is clearly too limited to assess adequate recovery. Do these findings question the validity of the TTM study? Certainly not. This RCT had a high-quality methodological design  and adequately compared the two TTM strategies. On the opposite, this meta-analysis was based only on retrospective studies , including patients with imbalanced characteristics at the baseline between TTM at 33 or 36 °C, with different quality of TTM  and relatively short-term outcome report . Moreover, the definition of UO was not standardized among studies and potentially not adequately assessed as in a prospective or randomized trial.Nevertheless, our results can at least cause some debate on how target temperature should be decided in the modern management of cardiac arrest patients. First, the application of the “TTM protocol”  might have been inadequately translated into clinical practice and resulted in more patients with early fever and poor temperature control. In one study including only OHCA with a shockable rhythm, patients treated with TTM at 36 °C spent less time outside the target temperature, with most of them having a greater probability to have at least one episode >38 °C ; this waUnfortunately, our results do not help to individualize target temperature to patients’ characteristics. After excluding patients with initial severe cerebral edema and early highly malignant patterns on electroencephalography (EEG), Callaway et al.  observedTTM is a relatively safe and effective strategy to improve the neurological outcome of cardiac arrest patients who remain comatose after hospital admission. Many questions on its effectiveness and implementation remain still unanswered; the translation of results from RCTs in clinical practice should be carefully evaluated as misinterpretation of such results might influence patients’ outcome. The use of TTM protocols aiming at avoiding poor temperature control is mandatory. However, the optimal approach to individualize target temperature based on patients’ characteristics remains still elusive. As for all newly commercialized drugs, epidemiological studies and surveillance programs with an adequate follow-up on large databases are necessary to understand how results from RCTs translate into medical practice.


TGFBI-associated corneal dystrophies are protein aggregation disorders in which the mutant TGFBIp aggregates and accumulates in the cornea, leading to a reduction in visual acuity and blindness in severe cases. Currently, the only therapy available is invasive and there is a known recurrence after surgery. In this study, we tested the inhibitory and amyloid dissociation properties of four osmolytes in an in-vitroTGFBI peptide aggregation model. The 23-amino acid long peptide (TGFBIp 611–633 with the mutation c.623 G>R) from the 4th FAS-1 domain of TGFBIp that rapidly forms amyloid fibrils was used in the study. Several biophysical methods like Thioflavin T (ThT) fluorescence, Circular Dichroism (CD), fluorescence microscopy and Transmission electron microscopy (TEM) were used to study the inhibitory and amyloid disaggregation properties of the four osmolytes . The osmolytes were effective in both inhibiting and disaggregating the amyloid fibrils derived from TGFBIp 611–633 c.623 G>R peptide. The osmolytes did not have an adverse toxic effect on cultured human corneal fibroblast cells and could potentially be a useful therapeutic strategy for patients with TGFBIp corneal dystrophies.Protein aggregation has been one of the leading triggers of various disease conditions, such as Alzheimer’s, Parkinson’s and other amyloidosis.  The irreversible pathological deposition of protein aggregates has been associated with diseases including Alzheimer’s, Parkinson’s and prion diseases5. Stromal corneal dystrophies associated with mutations in the Transforming Growth Factor-Beta Induced (TGFBI) gene, located on chromosome 5q318 are inherited protein aggregation disorders. They are characterized by age-dependent gradual accumulation and deposition of protein aggregates in various depths of the stroma leading to loss of corneal transparency and vision impairment5. The protein product of the TGFBI gene is a secreted 68-kDa extracellular matrix protein found in tissues throughout the body yet only the cornea is affected by the mutant proteins10.Intracellular and extracellular aggregation of proteins is often mediated by regions of low compositional complexity that may be intrinsically disordered in the native proteins10. The TGFBI gene has 74 mutations reported to date11, and the mutant protein is associated with modified protein stability, altered proteolytic processing, and deposition of insoluble aggregates in various layers of the cornea16. The aggregation and deposition of TGFBIp display different clinical phenotypes; the deposits range from amyloidogenic structures to amorphous granular deposits, or a combination of both17. Mutations in TGFBIp are not only known to alter the turnover rate but also alter the thermodynamic stability of the protein with several of the mutations leading to destabilized protein which is more likely to unfold20. The mutant protein also possesses different proteolytic processing and clearance mechanism in the eye compared to the wild type protein (WT). These proteolytic products can act as amyloid seeds that could trigger the TGFBIp aggregation pathway.TGFBIp consists of a secretory signal peptide, a domain rich in cysteine residues, a cell attachment tripeptide Arg-Gly-Asp (RGD) signal residue, and four Fasciclin 1 (FAS-1) domainsst and 4th FAS-1 domains of mutant TGFBIp have increased aggregation propensity compared to the WT24. We have also previously reported the protein composition of the amyloid fibrils from Lattice Corneal Dystrophy patients (LCD) using mass spectrometric assays25. The proteolytic fragments of TGFBIp derived from amyloid fibrils of patients, upon digestion with trypsin showed a higher abundance of peptides from the 4th FAS-1 domain of TGFBIp compared to healthy controls. Peptides G511DNRFSMLVAAIQSAGLTETLNR533, Y571HIGDEILVSGGIGALVR588, E611PVAEPDIMATNGVVHVITNVLQPPANRPQER642 and L497TPPMGTVMDVLKGDNRFSMLIt has been reported that the peptides derived from the 1533 were enriched in the patient samples compared to the controls26. The peptide region E611PVAEPDIMATNGVVHVITNVLQ633 26 has been associated with more than 16 clinically significant mutations, with high potential to form amyloid fibrils even under physiological conditions27. Eleven mutations in this peptide region are known to alter the overall net charge of TGFBIp26. Based on the in-vitro aggregation properties of peptides, TGFBIp 611–633 c.623 G>R and TGFBIp 611–633 c.622 N>K exhibited greater propensity to form amyloid fibrils and displayed remarkable resistance to thermal denaturation when compared to WT fibrils. The TGFBI c.623 G>R was first clinically reported by Gruenauer-Kloevekorn et al. in 2009 in two German families28.VAAIQSAGLTETLNR32. Osmolytes may be categorized as polyhydric alcohols, sugars (polyols), amino acids (and their derivatives), and methylammonium compounds35. Osmolytes are widely used to stabilize and facilitate protein folding since they can act as “chemical chaperones”37. Osmo-protectants and chemical chaperones have been shown to shift the equilibrium towards the native state33, by exhibiting a thermodynamic stabilization of the protein. This is accomplished by repopulating the denatured and native states, via unfavorable interactions with protein surfaces (a combination of backbone and side-chain interactions). Hence recently, more osmolytes are being evaluated as modes of treatment in various protein aggregation related disorders like Alzheimer’s disease and Huntington’s disease39.Osmolytes are small organic molecules, of diverse chemical structures, that regulate the solvent properties of cells, by conserving the native structures of proteins during an osmotic or thermal stress response611–633 with the mutation c.623 G>R (referred to as TGFBIp 611–633 G623R peptide in this manuscript). We chose four osmolytes Raffinose40, Taurine42, Betaine43 and Sarcosine43 .The purpose of this study was to investigate the role of osmolytes on the aggregation properties of model peptide TGFBIp Live-cell imaging -2, 5-Diphenyltetrazolium Bromide (MTT) Fig.  assays dTGFBI-peptide. The inhibitory effects of osmolytes were investigated further by fluorescence microscopy after staining with ThT  of osmolytes  for 24, 48 and 72 h and their ability to disaggregate the preformed amyloid fibrils were assessed as before Fig. . Treatme00 mM of 611–633 G623R peptide.The preformed amyloid fibrils from the peptide were treated with osmolytes and followed up to 72 h post-treatment and visualized under the fluorescence microscope by adding ThT dye Fig. . The res611–633 G623R peptide showed absorption minima around 218 nm and absorption maxima around 195 nm  and betaine (36% ± 5) suggesting disruption of the preformed amyloid fibrils, and corroborate with the ThT results. Among the osmolytes, raffinose and taurine displayed the highest disruption of the b-sheet structure when compared to betaine and sarcosine  by substitution with cationic residues resulted in a position-dependent alteration in the kinetics of amyloid formation26. Among these peptides, amyloid fibrils formed by TGFBIp 611–633 G623R peptide contained homogenous populations of β-sheet assemblies, displayed excellent thermal stability when compared to other peptides. Thus, we used TGFBIp 611–633 G623R as a model peptide for assessing the efficacy of various osmolytes on amyloid inhibition as well as disaggregation efficiency.The formation of insoluble amyloid aggregates is a pathological hallmark of 611–633 G623R rapidly aggregated to form amyloid aggregates in 24 h whereas incubation of peptides with an equimolar concentration of osmolytes resulted in a time-dependent decrease in amyloid formation. Preformed amyloid fibrils treated with osmolytes and monitored by ThT intensity, CD spectropolarimetry, fluorescence imaging, and TEM indicated Raffinose and taurine displayed significant dissolution of the amyloid fibrils. All observations confirm the effectiveness of osmolytes in the inhibition of amyloid fibril formation and the dissolution of amyloid fibrils. Among the four osmolytes tested raffinose and taurine have excellent amyloid inhibition as well as disaggregation properties.All the investigated osmolytes were non-cytotoxic to HCFs even at a final concentration of 1 M in the cell culture media. When incubated alone, the peptide TGFBIp et al. reported that the presence of preservatives such as benzalkonium chloride in eye drops close to the critical micellar concentration (0.001–0.02% (0.03–0.6 mM) accelerated the amyloid fibril formation in TGFBIp derived peptides and highlighted the potential risks associated with such formulations44 in eye drops. Use of several organic, polymeric, and inorganic nanoparticles, synthetic peptides, amino acids and sialic acid47 were reported to inhibit amyloid fibril formation. Palmal and Debnath et al., reported the utility of curcumin conjugated gold nanoparticles and epigallactocatechin-3-gallate conjugated polymer nanoparticles for inhibition and dissolution of preformed amyloid fibrils of Aβ1–40 peptide49. The strong affinity of nanoparticles for both oligomeric and fibrillar form of peptides was suggested to be responsible for stabilization of soluble oligomers which in turn attenuated the neurotoxicity of β-oligomers49. Unlike β-oligomers formed by Aβ1–40 peptide, the soluble oligomers of TGFBIp 611–633 c.623 G>R were non-toxic26. Thus, the dissolution of amyloid fibrils from TGFBIp by osmolytes may not have an adverse effect on the tissue and could potentially be a useful therapeutic strategy for patients with corneal dystrophies.There have been few previously reported studies on TGFBIp model peptides and the effect of chemical compounds on amyloid fibril formation and treatment options to disaggregate amyloid fibrils. Kato st FAS1 domain of TGFBI, Ozawa et al., have reported the use of a helium-cadmium laser with a power of 60–80 milliwatts at 37 °C under continuous agitation for disintegrating of amyloid fibrils50. Clinically, LASIK treatment in patients with corneal dystrophies worsens corneal haze and accelerated disease progression52. Byrne and Angell used protic ionic liquids for the disintegration of amyloid fibrils formed by hen egg-white lysozyme and the dissolved lysozyme retained substantial enzymatic activity53. A similar mechanism may hold good for the dissolution of TGFBIp amyloid fibrils by osmolytes. To the best of our knowledge, this is the first report on the use of non-cytotoxic osmolytes for the inhibition and disintegration of amyloid fibrils derived from TGFBI associated corneal dystrophies.Using a 22 residue model peptide derived from 1TGFBI gene, and to evaluate the pathologic role of the mutant protein56. All the generated animal models were not very successful to express the disease phenotype or in the survival of the animals. Since there are more than 65 mutations reported in this disease, it will not be feasible to generate animal models that represent each mutation or a universal model that can be used to study all the mutant phenotypes. Hence, the in-vitro peptide aggregation model will be more useful to study any treatment options that can be used to either prevent protein aggregation or dissolve preformed aggregates.There have been several previous attempts to generate a suitable transgenic animal model, either to knock-in or knock-out 611–633 c.623 G>R) with the amino acid change (Glycine to Arginine) was purchased from Synpeptide Co Ltd, Shanghai, China. The peptides were 95% homogenous as confirmed by reversed-phase high-performance liquid chromatography  in triplicates and images were captured with IncuCyte ZOOM System . Frames were then captured at 4-h intervals from 4 separate regions/well using a 10× objective for 20 h to observe cell toxicity.Cell viability of cultured human corneal fibroblast upon addition of osmolytes was assessed with Vybrant MTT Cell Proliferation Assay Kit . Culture media was replaced with 100 µl of PBS followed by the addition of 10 µl of 12 mM MTT solution. Cells were incubated in the dark at 37 °C for 3 hours before dissolving of formazan crystals with DMSO through the removal of all but 25 µl of medium and adding 50 µl of DMSO followed by thorough mixing and incubation at 37 °C for 10 minutes as per the manufacturer’s protocol. Absorbance was taken at 540 nm with a microplate reader (Tecan Infinite 200 PRO).611–633 c.623 G>R) from the 4th FAS1 domain of TGFBIp with the substitution, G623→R(EPVAEPDIMATNRVVHVITNVLQ) that rapidly formed amyloid fibrils was used in this study. The peptide was dissolved (0.6 mg/ml) in PBS and allowed to form amyloid fibrils in 50 ml Falcon tubes in a shaking incubator at 37 °C and 180 rpm with and without the addition of osmolytes.The 23-amino acid long peptide . The working concentration of all the osmolytes were 200 mM. The molecular weight of the various osmolytes used in the study is given in Supplementary Table 611–633 c.623 G>R peptide aggregation in the presence of various osmolytes at 24, 48 and 72 hours were measured. Dissolution of amyloid fibrils incubated with various osmolytes for 24, 48 and 72 hours after 24 hours of TGFBIp 611–633 G623R peptide fibrillation process in PBS buffer at pH 7.0 was collected in a Chirascan plus spectropolarimeter  using a 0.1 cm path length quartz cuvette. Spectra were recorded from 260 nm to 190 nm in 0.1 nm steps at a scan rate of 50 nm/min. The final spectrum was the average of three scans as per the manufacturer’s recommendation. The Mean Residual Weight (MRW) ellipticity ([θ]mrw) at wavelength λ was calculated using the equation:λ is the observed ellipticity at a particular wavelength (degrees), l is the path length (cm), and c is the concentration (g/ml). Secondary structures of the protein were analyzed using CDNN software.Far UV–CD spectra of the TGFBIp 218 values. Percentage inhibition for each osmolyte treatment per time point was calculated by normalizing [θ] 218 of osmolyte treatment with the [θ] 218 of untreated wells and expressed as a percentage.The percentage inhibition of secondary beta-pleated structure for inhibition and dissolution of fibrils of the peptide in the presence of osmolytes was calculated using [θ] 611–633 c.623 G>R peptide amyloid fibril formation by osmolytes and dissolving the preformed amyloid fibrils, the samples were incubated with 1:1 ratio of the peptide solution to ThT in the dark for 30 minutes. 25 µl of the samples were taken on to slides with coverslips and visualized under a fluorescent microscope . Three representative images from each time point under a given condition was used for the quantitation of fluorescence. Image J software was used to quantify the signal from the images and the values for each time point per osmolyte treatment were normalised to the untreated sample at that particular time point.For both inhibition of TGFBIp 611–633 c.623 G>R fibrils before and after treatment with osmolytes for 72 hours was investigated by Transmission electron microscopy (TEM) with a JEOL JEM-1010 transmission electron microscope using Digital Micrograph ™ 1.81.78 for GMS 1.8.0  at the Singhealth-core Electron Microscopy facility. 10 µl of amyloid fibril samples with and without osmolyte treatment were applied onto Formvar-carbon coated 300-mesh-size nickel grids. The samples were stained with Uranyl acetate solution, washed, dried, and observed at magnifications 8000–50000X at 80 kV.Morphological analysis of the TGFBIp p-value of less than 0.05 was achieved and very significant (**) with a p-value of less than 0.01 was achieved57.All numeric data obtained were expressed as mean ± standard deviation. For cytotoxicity studies, all the comparisons were done using one-way ANOVA followed by post-hoc Bonferroni test  for multiple comparisons. For both the experiments, values were deemed to be significant (*) when a Supplementary information.