These substantial fluxes are expected to increase as massive stocks of frozen organic matter in permafrost are liberated in a warming Arctic.Groundwater is projected to become an increasing source of freshwater and nutrients to the Arctic Ocean as permafrost thaws, yet few studies have quantified groundwater inputs to Arctic coastal waters under contemporary conditions. New measurements along the Alaska Beaufort Sea coast show that dissolved organic carbon and nitrogen (DOC and DON) concentrations in supra-permafrost groundwater (SPGW) near the land-sea interface are up to two orders of magnitude higher than in rivers. This dissolved organic matter (DOM) is sourced from readily leachable organic matter in surface soils and deeper centuries-to millennia-old soils that extend into thawing permafrost. SPGW delivers approximately 400–2100 m In this study, the authors show that water flowing through thawed soils below the tundra surface (supra-permafrost groundwater) can be a major source of dissolved organic matter (DOM) to Arctic coastal waters during the summer. This DOM contains leachates from old soil carbon stocks, including potential contributions from thawing permafrost. However, there is little information on direct groundwater nutrient inputs to the coastal ocean in the Arctic2. This is partly because of a perception that in northern high-latitude coastal regions permafrost constrains water to flow paths on the land surface. Supra-permafrost groundwater (SPGW) does, however, flow through seasonally thawed active layer soils during the summer and early fall4. Therefore SPGW has the potential to deliver appreciable quantities of terrestrially-derived nutrients to Arctic coastal waters. SPGW is the principal form of terrestrial groundwater entering nearshore coastal waters in the Arctic since sub-permafrost groundwater flow is firmly separated from the surface by several hundred meters of permafrost6.Groundwater is the largest active reservoir in the global hydrologic cycle and its movement from land to sea represents a major source of freshwater and nutrients for coastal ecological and biogeochemical processes8. Soils in northern high-latitude permafrost landscapes contain large amounts of organic matter with a high capacity to release dissolved organic matter (DOM) to aquatic systems9. Terrestrial DOM production and export is highest during the spring (May to June) when the thawed portion of the active layer is shallow and snowmelt-driven water flow is confined to near surface organic-rich soils and overlying plant litter layers12. DOM production and export is lower during the summer (July to October) when active layer thaw exposes deeper soil horizons and groundwater recharged from rainfall and melting ground ice saturates higher proportions of mineral soils14. Recent studies reveal that SPGW processes govern the summer transfer of DOM to streams and therefore influence riverine export to the coastal ocean17.In the Arctic, SPGW flow and nutrient transport from soils are tightly coupled because soil water interactions are largely confined to the shallow , but laterally extensive and highly permeable active layer2. Estimates of groundwater inputs to the coastal ocean are needed to support a more complete understanding of what fuels biological production and biogeochemical cycling in Arctic coastal waters. Climate change adds some urgency to this since warming is increasing groundwater discharge across circumpolar regions20, enhancing organic matter decomposition in the active layer, and liberating globally significant stores of organic matter held in high-latitude northern soils and permafrost25. A baseline understanding of how groundwater mobilizes organic matter held in coastal soils and permafrost is necessary for predicting responses to and feedbacks with climate change.In contrast to studies conducted on land, very few studies have focused on the role that SPGW plays in the direct transfer of DOM from land to the coastal ocean in the Arctic14C) dating. Lastly, we estimated DOM fluxes using concentrations of SPGW DOC and DON paired with groundwater discharge estimates that were derived from a steady-state excess radon (222Rn) mass balance model28 , from nearshore Arctic soils and quantifies inputs of SPGW DOM to coastal waters of the eastern Alaska Beaufort Sea Fig. . We firs29. We found a similar pattern in the release of DOM from these soil layers, where surface soils and thawed permafrost tend to have the highest yield (mg DOC and DON per gram of soil) and OC and ON normalized leaching potential (mg DOC and DON per gram of soil OC and ON)  because these soils have undergone more leaching events, greater cumulative effects of microbial mineralization with passing time, and are more stable because of mineral particle adsorption of DOM35. However, we found an increase in organic matter content farther down the soil profile, suggesting that cryoturbation , leaching, and/or decomposition processes concentrate organic matter near the permafrost boundary. Our results from frozen permafrost samples are consistent with other studies demonstrating that permafrost contains considerable amounts of readily leachable organic matter and releases high concentrations of DOC and DON upon thaw39. This is in part because permafrost can contain leachates and soils that are only partially decomposed; these soils have been well-preserved and frozen for millennia before thawing39. Overall, these trends are markedly similar to patterns in SOC and leachable soil DOC from coastal soils of Elson Lagoon near Utqiagvik (formerly known as Barrow) on the western side of the Alaska Beaufort Sea40. Convergence of our findings indicates that soils along the entire Alaska Beaufort Sea coast produce large amounts of leachable DOM that can be exported to lagoons during late summer.These observations are consistent with the notion that soils in northern high-latitude permafrost landscapes contain large amounts of leachable organic matter that can be exported to aquatic systems. Previous studies have demonstrated that DOM export is highest from plant litter layers and organic-rich soils near the surface (<5 cm) because SOM from these horizons have experienced less decomposition and fewer leaching events14C-DOC (radiocarbon dating of DOC) data reveals that SPGW DOC entering lagoons along the eastern Alaska Beaufort Sea coast is sourced from a combination of surface soils that contain freshly produced organic carbon and deeper soil horizons that may extend into thawing permafrost  near the land surface to ~5400 years before present  below the permafrost boundary , indicating that the fraction of readily leachable SOC is younger than the fraction of stable SOC in these deeper soil horizons with stronger mineral particle interactions. We found that groundwater has a 14C-DOC age of ~1300 yBP . The predicted Δ14C-DOC value of active layer-derived SPGW is –129 ± 30‰ or ~1040 yBP, which is younger (but within error) of the observed 14C-DOC age of SPGW. If shallow permafrost contributions are added in, then the expected Δ14C-DOC value is –153 ± 21‰ or ~1270 yBP, which is nearly identical to the observed 14C-DOC age of SPGW. Given that the 14C-SOC ages of the 30–40 cm and permafrost sections are markedly similar, these collective deeper soils represent a transient layer that likely experience a range of thaw conditions  near the permafrost-active layer boundary41. Although the assumptions in our calculations are simplified, this exercise reinforces the idea that a significant fraction of SPGW DOC is derived from deeper soil layers, including substantial contributions from the transient layer.Taking the analysis a step further, if it is assumed that SPGW receives DOC inputs from each soil horizon in proportion to their leachability . Our SPGW DOC concentration is over an order of magnitude higher than a recent estimate of the global average DOC concentration (2.7 mg C L−1) in groundwater aquifers from 15 countries and 4 continents at lower latitudes44.Measurements of DOC and DON demonstrate that SPGW supplies highly concentrated inputs of DOM to Arctic coastal waters during late summer Fig. . These c222Rn mass-balance model demonstrate that total groundwater discharge to Arctic lagoons is significant during the late summer  to Kaktovik Lagoon is an estimated 8.6 × 105 m3 day−1 or 42.6 m3 day−1 m−1. These values are similar in magnitude to previously reported values of 18 ± 10 Bq m−3 222Rn concentration and discharge of 12 ± 4 m3 day−1 m−1 in Elson Lagoon28.Results from a 1. Terrestrial-derived groundwater is typically a minor component of total groundwater contributions in the coastal ocean (1–10%), but much higher percentages  have been observed in coastal systems with strong topographic gradients at the land-sea interface and/or low wave and tidal-driven marine groundwater recirculation50. We expect that the percentage of terrestrial SPGW discharge in our lagoon system is in the lower range of previous estimates (<10%) because Kaktovik Lagoon is surrounded by flat tundra terrain in a region of continuous permafrost. However, terrestrial SPGW discharge is likely greater than 1% because tidal amplitudes and wave activity are very small. Therefore, assuming that 1 to 5% of the groundwater discharge that we measured with 222Rn in Kaktovik Lagoon is terrestrially-derived, we estimate that SPGW delivers roughly 8.6 × 103 to 4.3 × 104 m3 of freshwater day−1, 284 kg to 1420 kg of DOC day−1, and 17 to 86 kg of DON day−1 to Kaktovik Lagoon in late summer. This equates to approximately 400–210 m3 freshwater, 14–71 kg DOC, and 1–4 kg DON per km of shoreline per day in late summer . Although collective SPGW discharge accounts for only a small percentage of total riverine discharge from the North Slope in August (~1–5%), proportional contributions from SPGW DOM are potentially much more substantial (~14–70% for DOC and ~15–72% for DON) because of the large difference in groundwater versus river water DOC and DON concentrations during late summer. While the relative contributions of direct SPGW versus river inputs are probably minor in areas where major rivers, such as the Colville, flow into the Alaska Beaufort Sea, SPGW inputs may be the dominant source of freshwater, DOC, and DON along extensive stretches of coastline without sizable rivers.Results from the eastern Alaska Beaufort Sea coast provide strong evidence that SPGW can be a substantial source of DOC and DON to Arctic coastal waters. If we assume that SPGW fluxes are similar along the remainder of the Alaska Beaufort Sea coast, we estimate that direct SPGW fluxes range from 0.8–4.2 × 10In conclusion, contemporary SPGW fluxes supply substantial quantities of DOC and DON to coastal waters of the Alaska Beaufort Sea and thus represent an underappreciated source of energy for coastal ecosystems in the Arctic. The relative importance of SPGW DOM may be the greatest during late summer when river flow depreciates and groundwater discharge appreciates with maximum thawing of the active layer. The DOM found in SPGW during late summer is sourced from organic matter spanning the entire thawed soil profile, but primarily from highly leachable SOM near the surface and from deeper transient horizons. As warming continues in the Arctic, accelerated permafrost thaw and associated groundwater discharge have the potential to mobilize increasing amounts of soil organic matter.n = 10) and 8–12 August 2015 (n = 10), and then along Jago Lagoon on 17 August 2017 (n = 15)  on the landward sides of Kaktovik Lagoon on 9 August 2015 and Jago Lagoon on 8–9 August 2016 for bulk SOM measurements , which was used to buffer changes in pH and mimic the natural iconic strength of water in natural systems30. The wet soil: solution ratio of the leaching experiments ranged from ~1:10 to 1:4. The yields of DOC per g soil were similar between these soil: solution ratios in a study that took a more rapid leaching approach40. We expect no bias in our results related to differences in soil: solution ratios since our leaching experiments were conducted for a longer period of time. The beakers were covered with combusted aluminum foil and stored in a refrigerator at 4 °C. Beakers were shaken intermittently during a 24 h incubation period to simulate groundwater flow through the soil profile. Following, the soil water was filtered through a combusted glass filtration system holding a 0.7 μm glass fiber filter (precombusted at 450 °C >5 h). The filtrate was then dispensed into acid washed and Milli-Q rinsed polycarbonate bottles and stored frozen until DOC and DON concentration and 14C-DOC (radiocarbon of DOC) analysis. Bulk soils were stored in a drying oven (60 °C) for several weeks before they were finely ground using a mortar and pestle. Ground soil samples went through a vapor fumigation acid/base treatment step to remove inorganic carbon. This step involved storing soil samples in a vacuum-sealed desiccator in a drying oven (60 °C) with a beaker of concentrated HCl for 24 h. Afterwards, soil samples were removed and placed in another vacuum-sealed desiccator with a dish of NaOH pellets, and again stored in a drying oven at 60 °C for another 24 h. This latter step was conducted to neutralize any excess HCl that was not absorbed by the sample.Soil samples (>300 g frozen) were allowed to thaw in a refrigerator (4 °C) until the soils appeared moist, but not dripping with water (at most 24 h). Soils of the same type were then placed on combusted aluminum foil (550 °C for 1 h) and gently homogenized. Care was taken to maintain common soil features and to ensure the sample remained close to its natural condition. This also included removing large roots and anomalous material not representative of the soil horizon. Three subsamples were collected to obtain an average wet weight: dry weight ratio. Subsamples were dried in an oven at 60 °C for 24 h. Thawed soils were kept in the refrigerator for a total of 48 h before leaching. Sample wet weight: dry weight ratios were used to calculate the equivalent of 35 g of dry soil. Field-moist soils were then placed in a combusted glass beaker with 500 mL of a 0.001 N NaHCO3− and NH4+) was analyzed on a Seal-QuAAtro inorganic nutrient analyzer. Concentrations of DON were calculated as the difference between TDN and inorganic nitrogen. NO3− measurements made on the Seal-QuAAtro are the equivalent to NO3− plus NO2−. All 35 groundwater samples were measured for DOC, whereas the 20 samples collected in 2014 and 2015 were measured for DON (Supplementary Data n = 1), Jago (n = 1), and Okpilak (n = 2) rivers in ref. 43. In the case of the Okpilak River, two samples were averaged prior. Collective DOC and DON concentrations of north flowing rivers at their outlet from along the North Slope of Alaska were estimated using the average of data from the Turner (n = 1), Okpilak (n = 4), Hulahula (n = 1), Jago (n = 1), Canning (n = 1), Kuparuk (n = 5), Sagavanirktok (n = 5), and Colville (n = 3) rivers between July and the end of September 2006–2012 in ref. 43.Concentrations of DOC and total dissolved nitrogen (TDN) were determined using high-temperature oxidation performed on a Shimadzu TOC analyzer fitted with a total nitrogen module for chemiluminescence detection of nitrogen. Inorganic nitrogen 53. Sample water was diluted with pre-treated UV-oxidized nanopure water  and placed in a quartz reactor. The combined sample water plus treated nanopure water solution was acidified with 35 g of ultra-high purity (UHP), UV-treated full strength phosphoric acid and then purged with UHP nitrogen gas to remove any inorganic carbon. Pure UHP O2 was subsequently sparged through the system to provide an oxidant for the UV-oxidation of DOC. The sample was then oxidized with UV and the resulting CO2 was transferred to a vacuum line and cryogenically purified. Purified CO2 gas samples were converted to graphite targets by reducing CO2 with an iron catalyst under 1 atm H2 at 550 °C. Bulk soil samples were also high-temperature combusted using an Elementar vario EL Cube C/N analyzer. Bulk soil % OC and % ON were quantified during this step. The resulting CO2 was transferred to a vacuum line and cryogenically purified. The purified CO2 gas samples (soil C) were converted to graphite targets using a closed-tube Zn reduction CO2 graphitization method54. Targets were subsequently analyzed for stable and radiocarbon isotopes (δ13C‰ and 14C as fraction modern carbon). All Δ14C data (in ‰) were corrected for isotopic fraction using measured δ13C values that were quantified during the 14C-AMS procedure. We measured δ13C in these samples separately on a VG Prism Stable Mass Spectrometer at NOSAMS. Δ14C and radiocarbon age were determined from percent modern carbon using the year of sample analysis according to ref. 55.We analyzed 14C-DOC on a composite SPGW sample that was made from ten individual sample collections on the beach of Jago Lagoon in August 2017. The composite sample collected in 2017 had a 14C-DOC age of 1060 yBP, which is within error of the mean reported herein. This result was not used along with the individual measurements described in the previous paragraph to calculate an average SPGW age because it was determined using different methodology, but it does confirm that the average 14C-DOC age estimate used in this study is representative of August SPGW more generally. The groundwater composite collected in August 2017 was solid-phase extracted (SPE-DOM) using modified styrene divinyl benzene polymer PPL cartridges56. The SPE-DOM was high-temperature combusted using an Elementar el Vario Cube C/N analyzer and prepared using the same procedure described above. Although SPE-DOM using PPL cartridges typically recovers ~62% of the DOC sample as a salt-free extract56, we do not anticipate that the SPE-DOM process selectively concentrated compound classes that would result in a different 14C-DOC value than the UV-oxidation method.We also analyzed 222Rn measurements were made around Kaktovik Lagoon using a circuit of three RAD7 Radon-in-air monitors  connected to a RAD AQUA device following procedures outlined in ref. 27. The survey started after the system reached full gas/radioactive equilibrium. Measurements at ~1 m depth from the lagoon bottom were made in survey mode while driving a small inflatable boat around the lagoon. To optimize spatial resolution, the three RAD7 units analyzed samples staggered with 15-min intervals with water pumping at flow rates >6 L min−1. 222Rn concentrations shown at each location in Supplementary Figure 222Rn concentrations in discrete SPGW samples were collected using the Wat-250 ml protocol in the Durridge RAD7 user manual. 222Rn production measurements were conducted with dry lagoon sediments (ranging from 328 to 546 g) using a Durridge RAD7 bulk emissions chamber and methods outlined in the user manual.Boat-based 222Rn (t1/2 = 3.8 d) is a widely-used geochemical tracer of water flow through rocks and soil/sediments and thus serves a useful tool for quantifying submarine groundwater discharge1. Total groundwater discharge was calculated using a steady-state excess 222Rn mass balance model as described in ref. 27. We used a steady-state model because our lagoon system has no direct connections with the Beaufort Sea and has only two small (<3 m deep) and narrow (~12 m across) passes that connect with adjacent lagoons. It is important to note that there are several primary assumptions of the model: (1) lagoon 222Rn used for the calculation reflects the lagoon average concentrations over days to weeks; (2) the only significant 222Rn source is from the tundra active layer and lagoon benthic sediments, and does not include water that enters the lagoon via river inputs; and (3) the only losses of 222Rn are due to decay and atmospheric evasion. In addition, we assume marine inputs do not affect the 222Rn inventory in Kaktovik Lagoon since there is very little water exchange with the Beaufort Sea and lagoon water residence time is long during the summer (weeks to months) in comparison to the decay rate of 222Rn. The mass-balance model incorporates various end members that include data measured directly by this study, as well as data acquired elsewhere. Minor assumptions regarding this data are outlined in Supplementary Note 222Rn concentration was estimated from the average of three collected samples (223 ± 20 Bq m−3), while lagoon 222Rn was estimated from the average of data collected across two surveys (32.4 ± 3.85 Bq m−3). 222Rn production was measured from dry lagoon sediments and then converted to estimates of 222Rn in pore water (2932 ± 650 Bq m−3). Calculations regarding this conversion can be found in Supplementary Note 226Ra in this study, but rather used estimates from Elson Lagoon in ref. 28. Therefore we also assume that the 226Ra concentration between these lagoons is the same. Total groundwater 222Rn input was estimated using an iterative approach that accounts for the 222Rn flux from lagoon benthic sediments, atmospheric losses (using wind speed and water temperature data), and the introduction of 222Rn through the decay of 226Ra in the lagoon.Naturally occurring Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1


PVT1 locus as a previously unrecognized transcriptional regulator in CRC with a significantly high enhancer activity, which ultimately was responsible for regulating the expression of MYC oncogene. High expression of the PVT1 long-non-coding RNA (lncRNA) transcribed from the PVT1 locus was associated with poor survival among patients with stage II and III CRCs (p < 0.05). Aberrant methylation of the PVT1 locus inversely correlated with the reduced expression of the corresponding the PVT1 lncRNA, as well as MYC gene expression. Bioinformatic analyses of CRC-transcriptomes revealed that the PVT1 locus may also broadly impact the expression and function of other key genes within two key CRC-associated signaling pathways – the TGFβ/SMAD and Wnt/β-Catenin pathways. We conclude that the PVT1 is a novel oncogenic enhancer of MYC and its activity is controlled through epigenetic regulation mediated through aberrant methylation in CRC. Our findings also suggest that the PVT1 lncRNA expression is a promising prognostic biomarker and a potential therapeutic target in CRC.Accumulating evidence suggests that dysregulation of transcriptional enhancers plays a significant role in cancer pathogenesis. Herein, we performed a genome-wide discovery of enhancer elements in colorectal cancer (CRC). We identified The online version contains supplementary material available at 10.1186/s12943-020-01277-4. PVT1 locus as an epigenetic enhancer in CRC and provided a novel evidence for its role in specific targeting of the MYC oncogene. Furthermore, we found that PVT1 lncRNA was transcribed by the PVT1 locus via an enhancer-like activity. Multiple in silico datasets were utilized to evaluate the clinical significance of the PVT1 lncRNA. Pooling of seven such datasets improved the overall statistical power of the analysis and minimized potential cohort bias. Our analyses indicated that PVT1 is associated with cancer stemness and modulates key CRC-associated signaling pathways – the TGFβ/SMAD and Wnt/β-Catenin pathways. We additionally noted that high expression of the PVT1 lncRNA associated with poorer survival in CRC patients. Taken together, our data provide first evidence that the PVT1 locus plays a key role in CRC pathogenesis, and that it may serve as a prognostic biomarker and a potential therapeutic target in patients with CRC.Accumulating evidence indicates that the pathogenesis of colorectal cancer (CRC) is influenced by epigenetic modifications. Among these, in the past decade, alterations in enhancer elements have garnered a significant attention , and arePCAT1, CCAT1, CCAT2, PVT1 and MYC  genome browser. We discovered that the PVT1 locus indeed exhibited a strong H3K27ac enhancer signal, in a panel of cell lines , as well as in HCT116 CRC cells  assay in HCT116 cell lysates, we demonstrated that indeed the PVT1 locus formed a loop structure in a cis conformation with MYC  that targets the PVT1 lncRNA and permits its knockdown in the nucleus. Our approach led to successful knockdown of the PVT1 lncRNA in the cancer cell lines , with a simultaneous transcriptional suppression of the MYC mRNA  and humans (P = 0.011); suggesting that PVT1 lncRNA may either favor or serve as a marker of stemness which exists at the bottom of the crypt  CRC datasets , as well as with the MYC gene expression (ρ = − 0.3879, P = 0.0005, Fig. Aberrant methylation is one of the key regulators of enhancer activity in various genes. Interestingly, we observed a significant loss in CpG sequence methylation in the vicinity of the ter Fig. a. SpecifPVT1 lncRNA was reported as a stabilizer of the MYC protein [PVT1 locus may directly controls the MYC mRNA expression as an enhancer. Based on these data, targeting of the PVT1 lncRNA may be a potential therapeutic approach in CRC patients, which could eventually lead to suppression of the oncogenic MYC, at both the RNA and protein levels.Previously, the  protein . In addiAdditional file 1: Supplementary Table 1Additional file 2: Supplementary Table 2–7Additional file 3: Methods


A robust inflammatory response to tissue injury is a necessary part of the repair process but the deposition of scar tissue is a direct downstream consequence of this response in many tissues including the heart. Adult zebrafish not only possess the capacity to regenerate lost cardiomyocytes but also to remodel and resolve an extracellular scar within tissues such as the heart, but this scar resolution process remains poorly understood. This study aims to characterize the scarring and inflammatory responses to cardiac damage in adult zebrafish in full and investigate the role of different inflammatory subsets specifically in scarring and scar removal.tnfα+ and tnfα−) play prominent roles with manipulation of different phenotypes suggesting that pro-inflammatory (tnfα+) macrophages promote scar deposition following cardiac injury whereas tnfα− macrophages facilitate scar removal during regeneration. Detailed analysis of these specific macrophage subsets reveals crucial roles for Csf1ra in promoting pro-inflammatory macrophage-mediated scar deposition. Additionally, the multifunctional cytokine Osteopontin (Opn) (spp1) is important for initial scar deposition but also for resolution of the inflammatory response and in late-stage ventricular collagen remodelling.Using stable transgenic lines, whole organ imaging and genetic and pharmacological interventions, we demonstrate that multiple inflammatory cell lineages respond to cardiac injury in adult zebrafish. In particular, macrophage subsets (This study demonstrates the importance of a correctly balanced inflammatory response to facilitate scar deposition during repair but also to allow subsequent scar resolution, and full cardiac regeneration, to occur. We have identified Opn as having both pro-fibrotic but also potentially pro-regenerative roles in the adult zebrafish heart, driving Collagen deposition but also controlling inflammatory cell resolution.  Additionally, we investigate the role of Osteopontin (Opn), a multifunctional glycoprotein expressed by fibroblasts and inflammatory cells and a potent fibrotic factor in mammals,Here, we describe the timing, extent, and spatial distribution of the full inflammatory and scarring response to cardiac cryoinjury in a regenerative adult zebrafish model with a strong early recruitment of neutrophils followed by an influx of macrophages. Further investigation reveals two waves of macrophage populations, Details of transgenic lines and cardiac cryoinjury can be found in the In situ hybridization was performed using a commercial spp1 probe  and RNAscope® technology  on 4% PFA fixed TgBAC(spp1:mCherry) larvae and adult hearts following manufacturers protocols. Following in situ, samples were labelled with an anti-RFP  antibody using methods described in the Standard protocols were used for immunostaining, histology, and imaging of stable transgenic fluorescence. Details and antibodies used can be found in the Anaesthetized fish were intraperitoneally (IP) injected the day before cryoinjury (regime A) or at 3 days post-injury (dpi) (regime B) with 10 μL liposomes containing either clodronate or phosphate buffered saline (PBS) . For alteration of macrophage phenotype, anaesthetized zebrafish were IP injected with lipopolysaccharides (LPS)  or recombinant zebrafish IL10  immediately after cardiac cryoinjury.mpeg1:mCherry) were collected into PBS containing 10 mM HEPES, 30 mM taurine, and 5.5 mM glucose and cells dissociated by the addition of 0.25% trypsin, 12.5 M CaCl2, and 5 mg/mL Collagenase II . Dissociated cells were suspended in Leibovitz medium L-15 containing 0.3 mM glutamine (GIBCO), 0.8 mM CaCl2, Pen 50 U/mL/Strep 0.05 mg/mL, and 2% FCS. Mpeg1+ macrophages were sorted for mCherry expression on a BD Biosciences InFlux high-speed fluorescence activated cell sorter .Ventricles of wildtype Tg(spp1:mCherry)SN:374 reporter line, BAC recombination was performed as described.spp1 coding sequence was obtained  and the following primers were used to insert mCherry downstream of the spp1 start site (5′–3′): forward, tttagtttttctctctctgtttctcttctgttttagaatattttgcacacACCATGGTGAGCAAGGGCGAGGAG; reverse, ggtacacagaagactgtggcgacgaggagtgttaaaacaataatagatttTCAGAAGAACTCGTCAAGAAGGCG (lowercase indicates the BAC homology arms and uppercase indicates the mCherry cassette). To generate a spp1 mutant line a TALEN site was designed to target exon 7 of zebrafish spp1. TALEN plasmids were synthesized by ToolGen Genomics Toolmaker . The target site sequence was TACCACCATCATCCCAGTCACAGTCGATCCCACGCTGGGTCCCATTATCAACA, where TACCACCATCATCCCAGTCA represents the left TALEN and GCTGGGTCCCATTATCAACA represents the right. The red highlighted residue is deleted in the C331del line and the blue highlighted residues are deleted in the CGAT327-330del line. The TALEN plasmids were linearized, transcribed individually into TALEN mRNA using mMESSAGE mMACHINE T7 ULTRA kit (Ambion). Equal amounts (2 nL) of left and right TALEN mRNA were microinjected together into single-cell zebrafish embryos.To generate the TgBAC indicate that six animals are adequate for each parameter for statistical significance. This is also the number of fish used per time-point by others working on repair of the heart.mpeg1+ cells co-expressing tnfα determined manually. All GFP+ cells, regardless of intensity, were counted and all cells with clear outlines were counted in each image . Counts were taken from maximum projections of z-stacks to ensure maximum numbers of cells were analysed. Only cells where clear co-localization (e.g. by cell shape/outline) of both fluorophores was apparent were counted to prevent errors regarding overlap of cells. For all data sets a ROUT outlier test was performed and any significant outliers [Q  = 1%] were removed. Statistical significance was determined via non-parametric Mann–Whitney or Kruskal–Wallis/Dunn’s multiple comparison tests (details provided in figure legends). In all cases error bars represent SD. Indicated significance = *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001.In all cases All experiments were conducted with approval from the local ethical review committee at the University of Bristol and in accordance with UK Home Office regulations.Figure ,Figure ). In unwounded hearts a tissue resident population of L-plastin+ immune cells resided predominantly around the edge of the ventricle . L-plastin is a pan-leucocyte marker in zebrafish and mammals.,Cryoinjury to the adult zebrafish ventricle leads to repair with transient scarring that resolves over time . Following sham injury, no significant inflammatory or scarring responses were observed . By contrast, cryoinjury elicited a significant inflammatory response at 3 dpi, and a significant scarring response at 3 and 7 dpi . Inflammatory cell influx and scarring were significantly elevated in cryoinjured hearts compared to sham injury at 3 and 7 dpi . At 3 dpi, L-plastin+ cells are distributed throughout the myocardium, accumulating close to the injury site where considerable Collagen I deposition is observed . By 7 dpi, L-plastin+ cells are restricted almost exclusively to the injury site where significant Collagen I is still detected . At 14, 21, and 60 dpi both the inflammatory and scarring responses are reduced . Indeed, at 60 dpi the heart resembles that of an unwounded fish with the deposited Collagen I completely resolved .The scarring response was assessed by immunofluorescence analysis and subsequent quantification of Collagen I, suggested to be the major scar component.Figure S1). Collagen I staining generally gave a clearer result that was easier to quantify and less variable between different experiments. Interestingly, at 60 dpi (when scar tissue was only present in a few fish) Collagen I appears more restricted than collagen labelled with AFOG  suggesting a different composition of these rare late scars, supporting previous results suggesting that interstitial fibroblasts express collagens other than Collagen I at later time-points.Comparison of Collagen I and acid fuchsin orange G (AFOG) histological staining, which is commonly used to label scar collagen in injured hearts, on adjacent slides revealed similar distribution of collagens with both methods, particularly at early time-points . Neutrophils are generally considered to be the first responders to injurympx:GFP)i114 or Tg(lyz:DsRed2) transgenic zebrafish reveals rapid recruitment of neutrophils following cardiac cryoinjury, peaking at 6 hpi, but with almost complete resolution by 7 dpi . Although the vast majority of L-plastin+ cells at 6 hpi are neutrophils , analysis of Tg(gata2a:eGFP) transgenic fish reveals a significant recruitment of eosinophils to early zebrafish cardiac injury also . Analysis of double transgenic Tg(gata2a:eGFP); Tg(lyz:DsRed2) fish demonstrates two separate cell populations confirming the specificity of the Tg(gata2a:eGFP) fish for eosinophils .Previous reports have highlighted the importance of different innate and adaptive inflammatory cell populations following cardiac injury and have suggested that some may be required for cardiac regeneration in different species.mpeg1:mCherry) and Tg(c-fms:GFP) (csf1ra), revealed high numbers of macrophages present at all time-points . In unwounded hearts, analysis of L-plastin+, mpeg1+, and c-fms+ cell numbers suggests that almost all tissue resident, L-plastin+ cells are macrophages . Significantly elevated numbers of macrophages were present at 7 dpi corresponding to the commencement of resolution . This pattern is very similar to what has been reported for neonatal mice.mpeg1:mCherry) fish were analysed by FACS at different time-points post-injury  revealing an average of 0.5 ± 0.1% [unwounded (unw)], 1.2 ± 0.3% (1 dpi), and 7 ± 5% (3 dpi) mpeg1+ cells per fish .Analysis of two macrophage marker lines, Tg transgenic reporter. Significant numbers of lck+ cells were recruited to the damaged ventricle, peaking at 7 dpi  and were almost completely resolved by 14 dpi . Additionally, significant numbers of eosinophils were also observed at 7, 14, and 21 dpi . Interestingly, cumulative numbers of mpx+ (neutrophils), gata2a+ (eosinophils), mpeg1+ (macrophages), and lck+ (T-cells) present at each time-point does not notably differ from the total numbers of L-plastin+ cells suggesting that the cell types labelled by these transgenic markers represent the majority of all inflammatory cells responding to cardiac injury in adult zebrafish . Collectively, this analysis demonstrates that multiple different inflammatory cell types respond to cardiac injury in adult zebrafish and are present at similar time points and in similar ratios as observed in mammals. The main difference to adult mammals is the rapid attenuation of the inflammatory response between 7 and 14 dpi.Recent reports have suggested a potential role for T-cells in the response to tissue injuryc-fms:GFP) zebrafish received IP injections of clodronate or PBS containing liposomesFigure  (regime A)]. To assess the later function of macrophages (3–7 dpi), fish were first cryoinjured and then allowed to recover until 3 dpi when the peak inflammatory and scarring responses are occurring, and only then injected with clodronate or PBS liposomes, and here the repairing cardiac tissue collected at 7 dpi [Figure  (regime B)]. In both cases the number of c-fms+ cells was significantly reduced when compared to controls at 3 days post-treatment .As macrophages are the most abundant inflammatory cell type present in the heart at the crucial scar deposition and Collagen I regression time-points we sought to determine the role(s) of this population. To assess the early function of macrophages (up to 3 dpi), Tg reduced Collagen I deposition within the myocardium suggesting a role for macrophages in scar formation in line with previous reports , resulted in significantly more Collagen I at 7 dpi compared to control fish but no significant difference to control fish at 3 dpi, suggesting a failure in Collagen I resolution . These observations support previous reports from mammals that macrophages play a role in scar deposition.,Reducing the number of phagocytic cells during the early injury responses , to assess the activation state of the macrophages responding to cardiac injury . In unwounded fish, very few tnfα+ cells were observed whereas there was rapid accumulation of tnfα+ cells following cryoinjury with highly significant numbers observed at 1 and 3 dpi . The number of tnfα+ cells peaked with the early wave of macrophage influx at 3 dpi but reduced by 7 dpi when peak total macrophage numbers are observed and when scar resolution is commencing .Macrophages exist as a spectrum of different activated phenotypes ranging from pro-inflammatory through to anti-inflammatory/pro-resolution and these subpopulations can, at least partially, be defined by the expression of cytokines and other markers.mpeg1:mCherry); TgBAC(tnfα:GFP) double transgenic fish in more detail . In unwounded fish, very few tnfα+ macrophages were observed . Following cardiac cryoinjury, 91 ± 2% of mpeg1+ cells were also tnfα+ at 3 dpi . At 7 and 14 dpi, however, this number was significantly reduced . We next sought to determine if a more pro-inflammatory environment might influence the scarring response. Treating Tg(mpeg1:mCherry); TgBAC(tnfα:GFP) fish with a single dose of LPS at the time of injury resulted in significantly more mpeg1+ macrophages expressing tnfα at 7 and 14 dpi when compared to untreated fish , although the total number of macrophages was not significantly altered . Use of LPS to elicit a pro-inflammatory response has recently been validated in zebrafish.tnfα+ macrophages and scar deposition and that limiting the tnfα+ response is required for scar resolution to occur correctly. With these experiments we cannot completely rule out effects of other cell types .To further evaluate the potential opposing roles of these different macrophage populations, we analysed Tg zebrafish revealed significantly reduced numbers of mpeg1+ macrophages at 3 and 7 dpi across the whole ventricle . Numbers of mpeg1+ macrophages present only at the injury site in jcsf1ra4e1/j4e1; Tg(mpeg1:mCherry) fish, however, were similar to control fish suggesting that the remaining macrophages are functional and responsive to injury . Interestingly, analysis of jcsf1ra4e1/j4e1; Tg(mpeg1:mCherry); TgBAC(tnfα:GFP) fish revealed a significant reduction in the number of tnfα+ macrophages at the injury site at 3 dpi compared to wildtype  and these fish exhibited significantly reduced collagen deposition . LPS treatment of jcsf1ra4e1/j4e1; Tg(mpeg1:mCherry); TgBAC(tnfα:GFP) fish partially rescued the number of tnfα+ macrophages responding to cardiac injury  and this rescue was associated with significantly increased Collagen I deposition at 3 dpi suggesting a rescue of the scarring response also .We next assessed the inflammatory and scarring response to cardiac injury in mpeg1:mCherry); TgBAC(tnfα:GFP) fish with recombinant zfIL10, a cytokine that promotes anti-inflammatory polarization in macrophages, at the time of injury . This treatment reduced the number of tnfα+ macrophages at the injury at 3 dpi  and the amount of Collagen I, recapitulating the jcsf1ra4e1/j4e1 fish . We also determined the number of proliferating cells present in the ventricle of wildtype and jcsf1ra4e1/j4e1 fish at 7 dpi. No significant differences were observed . Collectively, our data suggest that an early pro-inflammatory response in macrophages, which requires Csf1ra, is necessary to initiate the scarring response in adult zebrafish but loss of csf1ra does not affect proliferation. Also, between 3 and 7 dpi, macrophages reduce tnfα expression and inhibiting this switch in phenotype with LPS treatment blocks scar resolution.To further address whether an initial macrophage pro-inflammatory response is required for scar deposition we treated wildtype Tg . Analysis of this reporter line reveals that mCherry expression recapitulates the previously described expression pattern of spp1 during development .spp1:mCherry); Tg(c-fms:GFP) fish reveals spp1 expression specifically at the site of cardiac injury at 3 dpi, partly co-localizing with Tg(c-fms:GFP) . In situ hybridization confirms the specificity of the TgBAC(spp1:mCherry) line in the craniofacial skeleton and the heart .One gene shown to be downstream of the injury inflammatory response and causal of fibrosis in mouse is spp1:mCherry); Tg(c-fms:GFP) fish demonstrates a subset of spp1+ c-fms+ macrophages following cardiac injury  with a significant increase at 7 dpi . Very few spp1+ c-fms+ cells are observed in unwounded hearts or at 1 dpi . At 3 dpi, another subset of spp1+ cells are c-fms− and are most likely interstitial fibroblasts ; ET37 fish suggests localization of spp1 in a subset of cardiac fibroblasts . The enhancer trap line, ET37, has been shown to label larval mesenchymal cells and we have further verified this line as labelling cardiac fibroblasts ; TgBAC(tnfα:GFP) fish at 3 and 7 dpi reveals double spp1+; tnfα+ and single positive cells suggesting dynamic expression changes of these two macrophage phenotype markers . To further identify the macrophages expressing spp1 following cardiac injury we treated TgBAC(spp1:mCherry); Tg(c-fms:GFP) fish with LPS at the time of injury  which significantly reduced the number of spp1+ macrophages suggesting these are more anti-inflammatory cells. Together, our data suggest that TgBAC(spp1:mCherry) fish recapitulate the developmental expression pattern of spp1, demonstrate expression in a subset of cell types in the heart, specifically after injury and that this expression is reduced with LPS treatment.Detailed analysis of TgBAC(spp1 mutant lines . These mutations result in frameshifts and a premature STOP codon that removes the majority of Opn functional domains predicted from mammals  although we cannot currently confirm a complete lack of protein in these fish. The resulting adult fish appear grossly normal . Analysis of the scarring phenotype in spp1−/− fish reveals a significant reduction in Collagen I deposition following cryoinjury at 3 and 7 dpi, similar to that described for Opn inhibition following skin wounding in mouse , suggesting that zebrafish Opn plays a role in scar resolution also. To assess effects of spp1 mutation on cardiomyocyte regeneration we assessed proliferation in wildtype and spp1−/− fish at 7 dpi . No significant differences were observed.To determine whether zebrafish Opn plays a role in regulating macrophage phenotype during cardiac repair/regeneration, we generated spp1−/− fish reveals significantly higher numbers compared to wildtype at 7 and 14 dpi  suggesting a failure in the resolution of the inflammatory response in the absence of Opn. Further analysis of spp1−/−; Tg(mpeg1:mCherry); TgBAC(tnfα:GFP) fish reveals increased numbers of tnfα+ macrophages at 3 and 7 dpi compared to wildtype fish  although normal numbers of total macrophages are observed at the injury site . This suggests that Opn is critical for controlling the switch in macrophage phenotype between 3 and 7 dpi from tnfα+ to tnfα− and that this is required for later resolution of inflammation and may impact on scar remodelling/resolution. Together, these data suggest bifunctional roles for Opn in both fibrosis/scar deposition, as in mammals, but also in regulation of inflammation and promotion of scar resolution, highlighting a previously unidentified role of Opn as a contributor to tissue regeneration.Quantification of L-plastin+ cells responding to cardiac injury in ,Cardiac scarring following tissue damage is a major contributing factor to the progression of heart failure, a leading cause of death in the western world. One major therapeutic goal is to improve the capacity of mammalian hearts to undergo regenerative rather than fibrotic repair. Interventions under investigation include generation of induced pluripotent stem cells (iPSCs) or differentiated cardiomyocytes and subsequent implantation into the infarcted region of the heart.,,,tnfα+ macrophages promote scar deposition, but a more anti-inflammatory phenotype is necessary to allow scar resolution. Csf1ra is required to maintain normal numbers of macrophages in the whole heart but the numbers responding to injury are relatively normal in jcsf1ra4e1/j4e1 mutants. However, these fish exhibit significantly reduced tnfα+ macrophages and scarring, supporting a role for these pro-inflammatory cells in promoting scar deposition, although the overall reduced numbers of cardiac macrophages may contribute to the reduced scarring observed in these fish. In vitro data suggests that human pro-inflammatory (‘M1’) macrophages are less fibrotic than alternatively activated (‘M2’) macrophage populations;The inflammatory response to tissue injury is a necessary consequence but one that drives subsequent fibrosis and scar formation.,,spp1+ cardiac macrophages at 7 dpi, a reciprocal expression pattern from that observed for tnfα. Studies in mammals have described contradictory roles for OPN in controlling macrophage phenotype; it may be a marker of alternatively activated ‘M2’ macrophages in the mouse hearttnfα and spp1 expression to define macrophage activation state, but further investigation will be required to delineate the precise phenotypes of macrophage populations in the adult zebrafish heart and to determine differences from mammalian macrophages, perhaps further illustrating why scar removal is so much more effective in zebrafish than in mammals.We have also identified a role for zebrafish Opn both in promoting scarring but also as a pro-regenerative factor in the heart. OPN is a multifunctional protein shown to be a pro-fibrotic factor downstream of the inflammatory response in mammals.spp1−/− fish exhibit reduced collagen deposition at 3 and 7 dpi but they exhibit significantly more scarring at 28 and 60 dpi when compared to wildtype fish, indicating a failure in resolution. Our data supports a role for Opn in specifically promoting Collagen I deposition, as observed in mammals, as this is the predominant collagen present at early stages post-injury. Interestingly, a recent report suggests collagens other than Collagen I are expressed during zebrafish cardiac fibrosis and scarring, particularly at later stages,spp1−/− fish exhibit an uncontrolled and expanded inflammatory response at 7 and 14 dpi and a concurrent increase in the number of tnfα+ macrophages, further supporting the role of Opn in control of the inflammatory response to cardiac injury. Analysis of proliferation rates in either the whole ventricle or specifically in Tropomyosin+ cardiomyocytes did not reveal significant differences between jcsf1rae1/jespp1−/−, and wildtype fish suggesting cardiomyocyte regenerative capacity is unaffected by the loss of csf1ra or spp1 and that this regenerative process is uncoupled from scarring and scar resolution. However, spp1−/− fish fail to completely remove scar tissue demonstrating that modulation of abnormal deposited scarring is a pre-requisite for complete tissue regeneration, regardless of cardiomyocyte proliferative ability.csf1ra is crucial for this early pro-inflammatory response and scarring. Subsequently, zebrafish macrophages exhibit a rapid phenotypic switch to reduce tnfα and increase spp1 expression and that this switch is necessary to allow inflammatory and scar resolution to occur. Our data provide evidence to suggest that the subtle regulation and balance of the inflammatory response and in particular of macrophage phenotypic state is crucial for scar resolution and subsequent full cardiac regeneration.Collectively, our data suggest that adult zebrafish possess an intricately balanced inflammatory response to cardiac injury, with similarities to that described in mammals. However, zebrafish utilize an initial pro-inflammatory response to drive Collagen I scar deposition rapidly following ventricular damage and we have demonstrated that ,Currently, curative treatments for severe cardiac scarring/heart failure include complex surgical interventions and, ultimately, a complete heart transplant. Therapeutic interventions that aim to improve the limited intrinsic regenerative potential of mammalian hearts or to reconstitute lost cardiomyocytes via exogenous stem cells or iPSCs are under intense investigation but have shown limited success, potentially due to the adverse scarred microenvironment these cells are placed into.cvz221_Supplementary_DataClick here for additional data file.


Deterministic assembly of large area van der Waals structures requires concrete indicators of successful interlayer coupling in bilayers grown by chemical vapor deposition. In this work, we examine the correlation between the stacking order and the interlayer coupling of valence states in both as-grown MoS2 homobilayer samples and in artificially stacked bilayers from monolayers, all grown by chemical vapor deposition. We show that hole delocalization over the bilayer is only allowed in 2H stacking and results in strong interlayer exciton absorption and also in a larger A-B exciton separation as compared to 3R bilayers. Comparing 2H and 3R reflectivity spectra allows to extract an interlayer coupling energy of about t⊥ = 49 meV. Beyond DFT calculations including excitonic effects confirm signatures of efficient interlayer coupling for 2H stacking in agreement with our experiments.Combining MoS 2 homobilayers as well as artificially stacked bilayers grown by chemical vapour deposition, and identify the interlayer exciton absorption and A-B exciton separation as indicators for interlayer coupling.The authors investigate the interplay between the stacking order and the interlayer coupling in MoS The band structure of TMD semiconductors is drastically modified by changing the sample thickness by just one atomic monolayer12. For instance, the combination of two different monolayer materials such as MoSe2–WSe2 into a heterobilayer results in type II band alignment and opens new research perspectives on periodic moiré potentials for carriers in the different layers and the resulting interlayer excitons17. Twisted homobilayers of graphene, WSe2, and MoSe2 allow accessing new superconducting phases and correlated insulating states20. To access the new functionalities provided by assembling monolayers to form multilayers it is necessary to identify physical parameters that strongly depend on interlayer coupling and to experimentally control them. One approach is to compare chemical vapor deposition (CVD)-grown MoS2 bilayers with artificially stacked bilayers made from CVD monolayers with 2H (180° twist angle) and 3R (0° twist angle) stacking. Studying these two precise alignments is also relevant for samples initially assembled with other twist angles as reconstruction results also in these experiments in the formation of μm-wide 2H and 3R areas22, which are energetically most stable. To artificially stack large area CVD layers and control interlayer coupling through stacking  is technologically relevant for 2D materials optoelectronics17, as CVD substrates are covered by a large number of monolayers and are very practical to stack (twist) due to their symmetric triangular shape and well-characterized edge termination.Transition metal dichalcogenides (TMDs) with the form MX25. This results in important changes in the optical spectra governed by K–K transitions as we observe strong absorption from interlayer excitons and a clear change in separation between A- to B-exciton transition in differential white light reflection at T = 4 K. These observations are made possible due to the drastically improved optical quality of CVD samples removed from the growth substrate and encapsulated in hBN26. We show that both indicators for interlayer coupling are absent in the measured 3R bilayer spectra as a hole hopping between the layers is symmetry forbidden27. Comparing for 3R (no interlayer coupling) and for 2H the A–B exciton absorption spectra allows us to extract an experimental value of the perpendicular hopping (coupling) term of t⊥ ≈ 49 meV, important for moiré superlattices28 and so far only roughly estimated from theory27.Here we show that the valence states for 2H bilayers are strongly impacted by interlayer coupling as the hole is delocalized over the two layersIn addition to our optical spectroscopy experiments we show in density functional theory (DFT) calculations, as well as by applying GW-type approaches, that the valence band (VB) splittings for 2H as compared to 3R are different due to interlayer coupling. In our calculated absorption spectra, including excitonic effects by solving the Bethe–Salpeter-equation (BSE) on top of GW calculations, for 2H stacking we show strong interlayer exciton absorption, absent for 3R stacking.30. In practice, most naturally occurring molybdenite shows 2H, not 3R stacking. In this work, we focus on high-quality CVD-grown flakes for several reasons: during CVD growth of MoS2 both 2H and 3R stackings for bilayers can occur31 and we are therefore able to compare the optical response for samples grown under identical conditions. Secondly, as all CVD flakes on our substrate show the same edge termination, we can artificially stack two layers in 2H and 3R configuration with a precise twist angle to compare with the as-grown samples—see “Discussion” below. Third, many monolayers cover the SiO2 substrate and can all be picked up in a single step, which makes fabrication of bilayer structures very efficient. CVD flakes with a larger surface area are also more convenient to fabricate devices with electrical contacts.The thermodynamically most stable configurations of TMD homobilayers are the 2H and the 3R stacking2/Si with 3R and 2H stacking are presented in Fig. 34. The SHG signal for 2H stacking was not detectable (inversion symmetry restored) but we could perform detailed angle-dependent SHG for the 3R stacking (broken inversion symmetry), see Supplementary Figs. 2 bilayers and monolayers were grown by a modified CVD process in which a Knudsen-type effusion cell is used for the delivery of sulfur precursor35. Using a water-assisted pick-up technique32, the as-grown CVD bilayers have been deterministically transferred and encapsulated in hBN to achieve high optical quality26, which has recently been shown to be crucial for optical spectroscopy on CVD samples lowering the typical emission linewidth from about 50 meV to below 5 meV at T = 4 K. An example of a photoluminescence spectrum of a CVD MoS2 monolayer used here is shown in Supplementary Fig. 24. After encapsulation, the samples were cooled down to T = 4 K in a closed-cycle cryostat and a series of differential reflectivity measurements with a home-built confocal microscope have been performed at different locations of the samples, see “Methods”. We define differential reflectivity as (Rsam − Rsub)/Rsub, where Rsam is the intensity reflection coefficient of the sample with the MoS2 layers and Rsub is the same structure without the MoS2. Note that the overall shape and amplitude of the differential reflectivity signal also depends on cavity effects (thin-layer interference) given by top and bottom hBN and SiO2 layer thickness (see ref. 36 for details). In Fig. 2 bilayers can be compared, see Supplementary Fig. 2 bilayers with 2H stacking37, in contrast to CVD-grown samples studied here. This observation of IEs is made possible by our specific CVD sample preparation for the optical spectroscopy experiment26. IE absorption was not detectable in very detailed earlier works due to considerably larger optical linewidth or detection of emission and not absorption41. In contrast to 2H stacking, in the 3R configuration no additional states are detected between the A- and B-excitons, thus indicating that the delocalization of holes is not allowed in this particular stacking order24, see below for a more detailed discussion. (ii) The separation between the A- and B-exciton transitions is considerably larger in the 2H bilayers (about 185 meV) as compared to the 3R bilayer . This is a second indication for efficient interlayer coupling of A–B valence states for 2H stacking, as the separation of the valence states mainly governs the A–B exciton separation42.Optical microscope images of as-grown CVD bilayers on SiO2 bilayers experience interlayer coupling resulting in interlayer exciton formation, here observed for a non-contaminated interface between the top and bottom layer. Contamination from secondary transfer processes could potentially suppress the coupling between the layers and hence IE formation. By choosing 2H or 3R orientation manually when stacking CVD-grown monolayers to form bilayers one can allow or disallow hole tunneling between the layers, respectively. This requires to pick-up the CVD-grown monolayers from their growth substrate while maintaining their structural integrity, optical quality, and a sufficiently clean interface after transfer. Furthermore, fine control of the twist angle between the top and bottom layer is needed, since the IE formation is allowed only in a precise stacking order, see sample preparation schematic in Fig. 32. First, CVD-grown monolayers have been carefully picked up from the growth substrate and transferred to polydimethylsiloxane (PDMS)  Fig. 32,43. ThDifferential reflectivity spectra have been collected from 10 different areas of the assembled 2H and 3R bilayers of Fig. 2 bilayers, see “Methods” for the computational details. Please note that our GW+BSE calculations are performed for MoS2 bilayers in vacuum for simplicity and not in hBN. The general target of our calculations is to understand the microscopic origin of the optical transitions and to reproduce the energetic order qualitatively. In GW calculations we compare band structures corrected by screening effects, the exciton description being added later. In the vicinity of the K-point of the Brillouin zone, see Fig. −1 states are clear. We find a VB splitting in the 2H bilayer that is 19 meV larger than in the 3R bilayer. By solving the BSE we obtain the absorption for the 2H and 3R bilayers shown in Fig. −1 states from distinct layers when they remain degenerate in 2H configuration. As a consequence, two distinct A-type excitons separated by only 14 meV constitute the A-peak of the 3R-bilayer absorption spectrum in our calculation, explaining its larger width compared to the 2H case, see Fig. 44. This is especially true for the VBs since the atomic orbitals that constitute those states are particularly sensitive to crystal field splitting.In addition to optical spectroscopy we perform beyond DFT calculations to study the striking differences between 2H and 3R MoSTo summarize the main experimental findings, first we observe strong interlayer exciton absorption between the main A- and B-exciton transitions for CVD-grown Fig.  and artit⊥ based on a k·p model of bilayers in the vicinity of K points28 and compare it to post-DFT estimates. As indicated in Fig. 3R is roughly given by the spin–orbit splitting as S3R = ΔSO. For 2H-stacking the A–B splitting in the valence band depends on the coupling energy t⊥ as 2H is the measured A–B exciton splitting of the as-grown 2H MoS2 bilayer and as a value for ΔSO we take the measured A–B separation in the 3R sample. For S2H = 183 meV and ΔSO = 155 meV, we obtain t⊥ ≈ 49 meV. This value can be compared to the ones extracted from our standard DFT calculations as previously done27, or from more advanced GW and GW+BSE calculations. Table 28 or Eq. 27, and experimental results is good as we reproduce the larger A–B splitting for the 2H bilayers as compared to 3R. Although we measure exciton transitions and not directly the VB splitting in the 2H bilayer, the agreement between theory and experiment strongly supports our interpretation for the reason behind the different A–B exciton splitting. It should be noted that for 3R bilayers, t⊥ = 0 since interlayer hopping is not allowed in this case. This short numerical analysis highlights that the efficiency of this interlayer coupling will depend on the ratio of t⊥ versus the spin–orbit VB splitting45, which is much smaller in MoS2 (ΔSO ≈ 150 meV) as compared to WSe2 (ΔSO ≈ 430 meV). This leads in principle to tunable interlayer coupling for MoS246 and so-called spin-layer-locking for WSe2 bilayers47.Our next target is to experimentally extract the interlayer hopping term, mentclass2pt{minim2 for 2H compared to 3R stacking has also been demonstrated in early experiments using optical transmission spectroscopy49 and more recently in angle-resolved electron emission spectroscopy50. The A–B exciton splitting in MoS2 bilayers has been previously studied by several groups54. In these reports, the spin–orbit coupling and interlayer coupling have been discussed, but neither interlayer exciton formation nor an experimental analysis of the coupling term t⊥. In our study, the direct comparison in the same set-up on the same substrate of 2H and 3R bilayers with good optical quality due to encapsulation allows to determine the difference in A–B exciton energies precisely, which we ascribe to interlayer coupling, as supported by our quasi-particle GW and absorption spectrum including excitonic effects calculations. From an experimental point of view, our results suggest two practical test criteria for interlayer coupling following artificial stacking: the strong interlayer exciton absorption and the clear difference in A–B exciton transition energies. The physics discussed here for 2H and 3R bilayers is also relevant for samples with a twist angle slightly different from 0° or 180° as reconstruction/self-rotation results in artificial stacks typically in large areas of 2H and 3R stacking, which will show the optical properties of the samples investigated here. Interestingly, interlayer exciton absorption has been reported in bulk 2H-MoTe2 and bulk 2H-MoSe256. Please see ref. 57 for related work on bilayer MoS2 .Larger A–B exciton splitting in bulk MoS2 crystals were grown on thermally oxidized silicon substrates  by a modified CVD growth method in which a Knudsen-type effusion cell is used for the delivery of sulfur precursor35.MoS2/Si substrate for hosting the van der Waals structure was cleaned with a 10-min ultrasonication bath in acetone and isopropanol followed by oxygen plasma exposure. A clean PDMS stamp was first placed on a glass slide and the SiO2/Si substrate containing the as-grown CVD MoS2 monolayers and homobilayers was brought in contact with the PDMS stamp32. The substrate was pressed against the PDMS stamp and distilled water droplets were injected at the perimeter of the substrate. Water droplets penetrated into the SiO2/MoS2/PDMS interface and after 1 min the SiO2/Si substrate was carefully lifted, resulting into the transfer of a large area of CVD-grown MoS2 triangles onto the PDMS stamp, as shown in Fig. 29 onto the target substrate and subsequent deterministic-dry transfer of the CVD-grown MoS2 triangles from the PDMS stamp on top of the hBN was applied. Thermal annealing at 150° for 30 min is performed after each transfer step.The SiOT = 4–300 K). The white light source for reflectivity is a halogen lamp with a stabilized power supply focussed initially on a pin-hole that is imaged on the sample. The emitted and/or reflected light  is dispersed in a spectrometer and detected by a Si-CCD camera. The excitation/detection spot diameter is  ≈1 μm, i.e., smaller than the typical size of the homobilayers.Low-temperature reflectance measurements were performed in a home-built micro-spectroscopy set-up assembled around a closed-cycle, low vibration attoDry cryostat with a temperature controller  hybrid functional65 has been used as an approximation of the exchange-correlation electronic term, including SOC, to determine eigenvalues and wave functions as input for the full-frequency-dependent GW calculations66 performed at the G0W0 level. An energy cutoff of 400 eV and a Gaussian smearing of 0.05 eV width have been chosen for partial occupancies, when a tight electronic minimization tolerance of 10−8 eV was set to determine with a good precision the corresponding derivative of the orbitals with respect to k needed in quasi-particle band structure calculations. The total number of states included in the GW procedure is set to 1280, in conjunction with an energy cutoff of 100 eV for the response function, after a careful check of the direct bandgap convergence . Band structures have been obtained after a Wannier interpolation procedure performed by the WANNIER90 program67. All optical excitonic transitions have been calculated by solving the Bethe–Salpeter equation69, using the 12 highest valence bands and the 16 lowest conduction bands to obtain eigenvalues and oscillator strengths on all systems. From these calculations, we report the absorbance values by using the imaginary part of the complex dielectric function.The atomic structures, the quasi-particle band structures, and optical spectra have been obtained from DFT calculations using the VASP packageSupplementary Information


Mobile health innovations are well adapted for ambulatory coronavirus disease 2019 (COVID-19) patients who risk clinical deterioration at home during the second week of illness.A short message service (SMS) communication program was implemented by French physicians to monitor COVID-19 patients after discharge from outpatient or emergency care. The aim of the SMS tracking is to advise patients about their need for medical reassessment if reporting worsening of COVID-19 symptoms.A follow-up via SMS to all confirmed positive patients in the Nîmes area (France) was established. Every morning, patients received four follow-up questions. Daily responses were converted to green, orange or red trees, analysed in real time by physicians. “Red” patients were called immediately to check their condition and organise transfer to hospital if needed. “Orange” patients were called within two hours to verify whether the specific instructions following the SMS had been followed.From March 21 to June 30, 2020, 1007 patients agreed to sign up to the SMS tracking, 62% were women and the mean age was 41.5 years (standard deviation (SD) 16.0). During follow-up, 649 (64%) became “orange” and 69 (7%) “red”. Ten patients were directly admitted to the Infectious Diseases Department during their follow-up due to clinical worsening, all but one as a result of SMS alerts and subsequent telephone assessment by physicians.SMS tracking platforms could be useful as an early warning system to refer patients with worsening clinical status to hospital-based care or additional clinician advice. The first non-imported coronavirus disease 2019 (COVID-19) cases were reported in France on February 24, 2020 ”.”“Question 1: Has your condition worsened since yesterday? (No (Send [1]), Moderately (Send [2]), A lot (Send [3])).Question 2: Do you have trouble breathing? (No (Send [1]), Moderately (Send [2]), Very much, while doing very little (Send [3]))Question 3: Can you easily manage your daily life ? (Yes (Send [1]), No, not at all (Send [2]))Question 4: Do you feel able to continue home confinement? (Yes (Send [1]), No, not at all (Send [2]))Every morning at 08:45, patients received the following questions:Questions were chosen to be easily understandable and to cover the different aspects of COVID-19 symptoms and the related isolation . The questionnaire took less than 5 min per day to complete.An automated reminder was sent if no response was received by 12:00. The tracking platform generated a web page with each row representing a patient integrated in a separate dashboard distinct from patients’ medical chart. Daily responses were converted to green, orange or red trees, analysed daily in real time from 08:00 to 20:00. One infectious diseases physician dedicated 50% of their working hours to the inclusion of patients, colour checking and contacting the orange/red patients.Patients with only “1” responses were displayed in green and received the following message: “Thank you for participating. Your condition is stable or improving. Follow-up continues at home. Continue to adhere to strict containment guidelines. See you tomorrow.”If there was at least one “2” response but no “3”, patients were displayed in orange and the following message was sent: “Thank you for participating. It seems to us that your condition requires re-evaluation by your general practitioner today to continue follow-up at home. Please continue to adhere to the strict containment guidelines. See you tomorrow.”If there was at least one “3” response, patients were displayed in red and the following message was sent: “Please dial 15 [emergency medical assistance service] promptly so that your condition can be assessed by an emergency and/or infectious diseases specialist.”“Red” patients were called immediately to check the patient’s condition and organise transfer to hospital if needed. “Orange” patients were called within 2 h to see if the specific instructions following the SMS had been followed.We were rapidly able to offer the inclusion of patients into the tracking platform to nine other ambulatory COVID-19 centres in the Gard department: two secondary general hospitals and seven general practitioner groups caring for suspected and confirmed COVID-19 patients.General practitioners were informed of the existence of the SMS tracking system and how to join via e-mail from the ARS .From March 21 to June 30, 2020, five patients  refused to be included and 1007 patients agreed to sign up to the SMS tracking, 62% were women and the mean age was 41.5 years (standard deviation (SD) 16.0). Of these, 994 (99%) had at least 1 day of follow-up and 850 (85%) more than 5 days of follow-up. The mean length of follow-up was 13.2 days (SD 5.4).Figure During follow-up, 649 (64%) became “orange” and 69 (7%) “red”, of whom 30 progressed from “orange” to “red” and 39 from “green” to “red”. We found no differences in age and sex between the 688 patients who became red or orange  and those who stayed green  .The main limitation of our study is that the impact assessment of our SMS tracking platform was not possible because we were unable to create a control group due to the high participation rate and the early launch of the platform in the epidemic, without data for the cases that occurred prior to the launch.Mobile health interventions, including SMS, increase access to care and enhance the efficiency of health service delivery in different conditions –5. PatieMost mobile health interventions in the context of COVID-19 have been developed for screening patients  or tWe share here one example of a physician-monitored SMS to connect with patients with COVID-19 after discharge from outpatient or emergency care that was quickly developed and highly acceptable to patients and clinicians. SMS tracking platforms could be useful as an early warning system to refer patients with worsening clinical status to hospital-based care or additional clinician advice.


In recent years, metabarcoding has become a key tool to describe microbial communities from natural and artificial environments. Thanks to its high throughput nature, metabarcoding efficiently explores microbial biodiversity under different conditions. It can be performed on environmental (e)DNA to describe so-called total microbial community, or from environmental (e)RNA to describe active microbial community. As opposed to total microbial communities, active ones exclude dead or dormant organisms. For what concerns Fungi, which are mostly filamentous microorganisms, the relationship between DNA-based  and RNA-based (active) communities is unclear. In the present study, we evaluated the consequences of performing metabarcoding on both soil and wood-extracted eDNA and eRNA to delineate molecular operational taxonomic units (MOTUs) and differentiate fungal communities according to the environment they originate from. DNA and RNA-based communities differed not only in their taxonomic composition, but also in the relative abundances of several functional guilds. From a taxonomic perspective, we showed that several higher taxa are globally more represented in either “active” or “total” microbial communities. We also observed that delineation of MOTUs based on their co-occurrence among DNA and RNA sequences highlighted differences between the studied habitats that were overlooked when all MOTUs were considered, including those identified exclusively by eDNA sequences. We conclude that metabarcoding on eRNA provides original functional information on the specific roles of several taxonomic or functional groups that would not have been revealed using eDNA alone. Metabarcoding, i.e. the combined use of universal DNA barcodes and high-throughput sequencing, is now a standard approach to characterize microbial communities from nucleic acids directly extracted from environmental samples  . This stAt the global scale, the monitoring of fungal diversity by metabarcoding has shown how it is shaped by a wide set of environmental factors including climate , seasonsFor these reasons, high-throughput profiling of fungal communities has been suggested as a method to monitor forest and soil health, assuming that local fungal diversity is directly linked to ecosystem functions  such as In fungal taxonomy and community ecology, the most studied DNA barcode is the nuclear ribosomal RNA (rRNA) intergenic spacer (ITS) locus –20. ThisReports of microbial, either prokaryotic or eukaryotic, community metabarcoding on both eDNA and eRNA highlighted either a strong correlation between “active” (RNA-based) and “total" (DNA-based) microbial communities –29, or oIn metabarcoding studies, it is essential to identify and eliminate artefactual sequences and spurious taxa whose presence may interfere in data analysis and mask or on the contrary exacerbate differences between datasets . ArtefacThe aim of the present study was to compare the performance of different metabarcoding sequence datasets, generated from eDNA and eRNA, for the discrimination of fungal communities collected in different habitats at a regional scale in Northern Italy. We sampled three contrasted habitats (or substrates), namely decaying wood, forest and grassland soils. In mycology it is widely accepted, as reflected by fungal floras and field guides , 50, 51,The four study sites  are locaIn both grassland and forest plots, 20 soil cores  were regularly collected along two distinct 20 m-long linear transects. After litter and plant removal, each sample was sieved (2 mm mesh size) and all samples from the same grassland/forest plot were mixed together in equal amounts to constitute a single composite sample that was frozen in liquid nitrogen and stored at -75°C before DNA/RNA co-extraction. About 100 pieces of decomposing wood were regularly collected in the vicinity of the two transects used for forest soil collection. Wood samples (lying on the ground or not) represented different size classes (from twigs to trunk fragments), stages of decomposition and the different tree species present on the sampling site. After removing bark fragments, wood was reduced to sawdust using a sterilized stainless steel grater. For each forest, all samples were mixed together in equal amounts, frozen in liquid nitrogen and stored at -75°C until RNA/DNA extraction.260:OD280 ratio, Nanodrop ND-1000 spectrophotometer, Thermo Fisher Scientific, Waltham, MA, USA) and quantified by fluorimetry using the Qubit dsDNA BR Assay kit and Qubit Fluorimeter 2.0 (Thermo Fisher Scientific).Soil RNA was extracted from 2 g of material using the RNA Power Soil extraction kit from MOBIO laboratories  according to the manufacturer’s instructions. Soil DNA was co-extracted using the PowerSoil DNA Elution kit (MOBIO laboratories). Wood RNA and DNA were co-extracted from 100 mg of wood following the protocol described by Adamo et al., . Purity Five hundred ng of soil/wood RNA were used for cDNA synthesis in the presence of 4 μμmol of random hexamers . The mixture was first heated 5 min at 70°C and kept on ice for at least two min before adding 10 μμl of a reaction mixture comprising 4 μμl of a 5x buffer (Thermo Fisher Scientific); 2 μμl of a 10 μμmol dNTP solution; 1.5 μμl RNAsin RNase inhibitor at 40 U/μμl; 2 μμl of 5% Bovine Serum Albumin (BSA); 1 μμl of M-MLV Reverse Transcriptase at 200 U/μμl (Thermo Fisher Scientific) and 0.5 μμl of RNA grade water. After 1 h at 42°2°C, the enzyme was inactivated by incubating 10 min at 70°C.Amplifications of fungal ITS2 sequences were performed following a nested PCR approach using as starting material either 20 ng of environmental DNA or 1 μμl of cDNA solution. The nested PCR approach was adopted to avoid the artefactual amplification of plant sequences likely to be present in the samples (soil and decaying wood), but also because the direct use of the fITS9—ITS4 primer pair on environmental nucleic acids failed to amplify the ITS2 region from several samples . InAll amplifications were performed in a final volume of 25 μμl comprising 2.5 μμl of a 10 x Taq buffer (Thermo Fisher Scientific), 0.1 mmol dNTPs; 2 μμmol of forward and reverse primers; 0.3% of BSA; 1U of Taq DNA polymerase (Thermo Fisher Scientific); the appropriate amount of DNA or cDNA and ultrapure water. For the ITS1F—ITS4 primer pair, after an initial denaturation of 5 min at 95°C, amplification proceeded through 35 cycles of 30 s at 94°C, 45 s at 54°C and 1 min at 72°C. After a final extension for 10 min at 72°C, 1 μμl of each PCR product was used as template in the nested PCR reaction with primers fITS9 and ITS4. After an initial denaturation of 30 sec at 98°C, amplification proceeded through 30 cycles of 10 s at 98°C, 30 s at 64°C and 20 s at 72°C, followed by a 10 min extension at 72°C. All PCRs were performed in a T3000 thermal cycler .PCR products were controlled by electrophoresis on 1% agarose gels, and the four independent PCR amplifications performed on each DNA/cDNA extract were pooled in equal amounts and purified using the Wizard SV Gel and PCR Clean-Up System (Promega) following the manufacturer’s instructions. After purification the PCR products were quantified using the Qubit dsDNA BR Assay kit and Qubit Fluorimeter 2.0 (Thermo Fisher Scientific) in order to prepare libraries for a paired-end sequencing (2x250 bp) with the Illumina MiSeq technology by Fasteris .Bioinformatics analyses were performed essentially as described in Voyron et al., . Paired-http://unite.ut.ee, last accessed on May 12 2017). OTU functional annotation made use essentially of the FUNGuild database : [No. of DNA reads] (log2(RNA:DNA+1)) was computed and plotted on a horizontal axis for each of the five most represented fungal classes and for habitat. Symbol size is proportional to the relative abundance (average reads number among the samples) of the taxa in the dataset. For the Agaricomycetes we distinguished symbiotic  MOTUs (symb.) from saprotrophic and undefined ones (non-symb). Black circles give the mean values and black bars the standard deviations for each fungal class. Identical red letters on the left  indicate which of the distributions are statistically similar .For each MOTU (grey/orange circles) the log(TIFF)Click here for additional data file.S2 Fig2 of the ([No. of RNA reads]: [No. of DNA reads] +1) ratio was calculated for each of the individual sample in which the taxon was present. Bars that give the standard deviation of the mean illustrate that for several of the taxa their relative abundance as DNA or RNA reads varied considerably between samples.Log(TIFF)Click here for additional data file.


More importantly, we have improved phase singularity detection with the Goos–Hänchen (GH) effect. The GH shift is known to be closely related to optical phase signal changes; it is much more sensitive and sharp than the phase signal in the plasmonic condition, while the experimental measurement setup is much more compact than that of the commonly used interferometer scheme to exact the phase signals. Here, we have demonstrated that plasmonic sensitivity can reach a record-high value of 1.2862 × 109 µm/RIU with the optimum configurations for the plasmonic metasensors. The phase singularity-induced GH shift is more than three orders of magnitude larger than those achievable in other metamaterial schemes, including Ag/TiO2 hyperbolic multilayer metamaterials (HMMs), metal–insulator–metal (MIM) multilayer waveguides with plasmon-induced transparency (PIT), and metasurface devices with a large phase gradient. GH sensitivity has been improved by more than 106 times with the atomically thin perovskite metasurfaces (1.2862 × 109 µm/RIU) than those without (918.9167 µm/RIU). The atomically thin perovskite nanomaterials with high absorption rates enable precise tuning of the depth of the plasmonic resonance dip. As such, one can optimize the structure to reach near zero-reflection at the resonance angle and the associated sharp phase singularity, which leads to a strongly enhanced GH lateral shift at the sensor interface. By integrating the 2D perovskite nanolayer into a metasurface structure, a strong localized electric field enhancement can be realized and GH sensitivity was further improved to 1.5458 × 109 µm/RIU. We believe that this enhanced electric field together with the significantly improved GH shift would enable single molecular or even submolecular detection for hard-to-identify chemical and biological markers, including single nucleotide mismatch in the DNA sequence, toxic heavy metal ions, and tumor necrosis factor-α (TNFα). In this work, we have designed highly sensitive plasmonic metasensors based on atomically thin perovskite nanomaterials with a detection limit up to 10 In the past decade, biosensors have provided an effective way for improving quality of life ,2,3. Bio−15 mol/L) in complex matrices such as urine, saliva, and blood serum. Plasmonic sensors or surface plasmon resonance (SPR) sensors are one of the most commonly-used optical sensing devices for real-time monitoring of chemical and biomolecular interactions ,21,22,23Recently, researchers have discovered that the use of optical phase singularity could be a powerful solution to address these two challenges ,29,30. P4H9NH3)2PbBr4) by the solution phase growth method. They tuned the thickness of the perovskite layers from a single layer to 3 layers, 8 layers and 22 layers. The corresponding photoluminescence emission peaks are shifted with the changing band gap for these thin perovskite layers. Later, Bao et al. [3NH3PbX3  perovskite with broadband photoluminescence emission ranging from 660 to 730 nm and a high photoresponsivity of 22 AW−1 under the excitation of 532 nm laser sources. The rapid development of the fabrication techniques for different types of nanomaterials has allowed many breakthroughs in the optical and electronics properties, e.g., high charge carrier mobility, negative refraction, hyperbolic dispersion ,37,38,39o et al.  reported−10 refractive index units (RIU) for the target sample solutions. More importantly, we have improved the phase singularity detection with the Goos–Hänchen (GH) effect. The GH shift is known to be closely related to optical phase signal changes; it is much more sensitive and more sharp than the phase signal in the plasmonic condition, while the experimental measurement setup is much more compact than that of the common used interferometer scheme to exact the phase signals. Here, we have demonstrated plasmonic sensors with an ultra-high value of 1.2862 × 109 µm/RIU with the optimum configurations for the plasmonic metasensors. For the first time, GH shift generated through the phase singularity based on the 2D perovskite materials is more than three orders of magnitude compared to other metamaterials schemes, including Ag/TiO2 hyperbolic multilayer metamaterial (HMM) [9 µm/RIU) than those without them (918.9167 µm/RIU). In this paper, we have designed ultrasensitive plasmonic biosensors with the integration of atomically thin perovskite nanomaterials on metasurfaces reaching a detection limit up to 10al (HMM) , a metalal (HMM) , a metasal (HMM)  and evenal (HMM) . The plaTo evaluate the sensing performances of the 2D perovskite-enhanced plasmonic configurations, we have chosen an established Kretschmann excitation method as the standard modeling of the sensor head, as shown in In this prism-coupling method for plasmonic excitation, we used a high refractive index equilateral SF11 prism. The refractive index of the prism is obtained by the equation below :(1)npriseference . The thieference  and fixeeference ,59. The eference ,61: (2)nbio, ranging from 10−8 refractive index units (RIU) to 10−1 RIU [The dielectric constants of perovskites that are used for the plasmonic modeling extracted from experimental results are shown in 10−1 RIU ,62,63. TSPR), or the change in resonance angle (∆θSPR) and the change in resonance wavelength (∆λSPR). However, these detection methods are not able to achieve the sensitivity for tiny refractive index changes below 10−5 refractive index units (RIU). Recently, the phase interrogation approach has been exploited to significantly improve the sensitivity of the SPR sensors. Experimental schemes for differential phase measurements based on interferometric and ellipsometric configurations have been demonstrated to extract the phase signal (ϕp) of the reflected light. The phase signal led to a much sharper change to the surrounding media at the resonance interface in comparison to the change in the other parameters of reflected light mentioned above. Here, the Goos–Hänchen (GH) shift (Lshift) emerged as a higher order signal change of the phase, and corresponds to the lateral shift of the reflected beam at the sensing substrate. This lateral shift in the direction along the propagation direction of the surface plasmon waves (SPWs) is similar to the Goos–Hänchen (GH) shift (Lshift) that is usually observed when an abrupt phase change is induced for phenomena such as the Berry phase, Brewster’s angle phenomenon and the Aharonov–Bohm effect. However, the quantitative measurements of the Goos–Hänchen (GH) shift (Lshift) for these phenomena are challenging due to the few micrometers magnitude shift in general. Under strong surface plasmon resonance effects, this shift could be optimized and enhanced to 2 orders of magnitude larger. It is known that for conventional surface plasmon resonance (SPR) sensors, detection merits are mainly based on the scanning of the change in reflected light intensity (∆RWe have used the Transfer Matrix method (TMM) with Fresnel equations to do systematic investigation on our 2D perovskite-enhanced plasmonic models. The tangential part of the electromagnetic field for the sensing substrate from the 1st layer to the last layer are followed the boundary condition, as below: For the p-polarization light, the Transfer Matrix (TM) for a multilayer model is described as: The Fresnel reflection coefficient for p-polarized light is calculated by:shiftL) of this multilayer model are obtained as [Thus, the phase (ained as :(8)ϕp=arshiftL). The phase (pϕ) and Goos–Hänchen (GH) shift (shiftL) are very sensitive to the refractive index change When the surface plasmon resonance is excited, the value of the reflected light intensity is close to zero, and a large phase jump is observed at the SPR angle. The phase retardation at this singular point leads to a large Goos–Hänchen (GH) shift (The SPR sensitivity based on Goos–Hänchen (GH) shift is defined as :(10)SGH=ε⊥) and longitudinal directions (ε∥), as below: N = 1 – mW/mP is the plasmonic concentration for the metallic nanomaterials inside the structures, and mW and mP are widths and periodicity of the metasurface nanostructure. This equation is applicable for both metal array and groove designs [The design of the metasurfaces are studied based on the effective medium theory (EMT) method. It is known to be used for the optimization of plasmonic nanorod arrays and groove structures. Here, we can obtain the dielectric constant for the periodical array structures respectively for the metasurfaces along the transverse ( designs . In addishift) for the refractive index change (∆ntarget) induced by the target sample solutions at the sensing interface. One of the prominent physical features for surface plasmon resonance (SPR) is that under the resonance condition, the electric field excited at the substrate would be largely enhanced due to the resonant coupling of the light energy. The maximum electric field intensity can be two orders of magnitude higher than the initial field value of the incident light. As a result, the energy level dissipated to the dielectric layer is correspondingly larger than the total internal reflection (TIR) phenomena with only dielectricity and without the use of the metallic thin films. The resonance energy of the evanescent field penetrating to the surrounding media led to a much increased lateral shift from a few micrometers less than 10 µm (<10 µm) for normal TIR to more than 60 µm (>60 µm) for plasmonic resonance. The Goos–Hänchen (GH) shift can be quantitatively calculated from Equation (9) and is proportional to the phase retardation (∆ϕp), occurring at the sensing substrate. In Based on Equation (10), the SPR sensitivity based on Goos–Hänchen (GH) shift for the plasmonic metasurface structures is strongly related to the lateral shift of the p-polarization light (L−12), picomoles/liter was fixed to be ∆nbio = 1.2 × 10−6 RIU. Here, we have observed several consistent characteristics for the 2D perovskite-based plasmonic sensing response curves  and the resonance dip angle shift (∆θSPR = 1.7484°) from −3 RIU and 1.2 × 10−1 RIU with the GH shift change (∆Lshift = 1543.5 µm) in bio = 1.2 × 10−6 RIU. The resonance dip angle shift increases with the increased perovskite thickness and is not affected by the resonance depth during the sensing process. However, the slightly improved angular SPR sensitivity is significantly compromised by the much more wide plasmonic resonance curves. −1 RIU change. For the ∆nbio = 1.2 × 10−6 RIU, the highest angular and phase signal changes with 2D FAPbI3 perovskite are respectively 0.00001° and 4.6954°. These signal changes are much lower than the device resolution for some of the rotation stages and phase detectors. Aa a result, the conventional angular scanning and phase interrogation methods are not competent for the detecting of the tiny refractive index change up to 10−6 RIU in comparison to the large GH shift of ~1600 µm with 2D FAPbI3 perovskite-enhanced plasmonic device. For the 2D MAPbI3−xClx perovskite structures, the highest GH sensitivity is 4.0518 × 108 µm/RIU, with optimum configuration of 48 nm silver thickness and single layer perovskite  with a lateral shift of 0.0028 µm. The capability to detect the small refractive index change promised the sensing of low-concentration chemical and biological molecules up to 1 fM (10−15 mol/L) and even 1 aM (10−18 mol/L). One can choose the type of perovskite configurations based on the molecular weight and also the concentration range. To achieve wide dynamic detection range, we can use the tunable laser to change the excitation wavelength for continuous sensing of both small and large target analytes with different concentrations. In shift = 17.3576 µm is shown, corresponding to the refractive index induced by the target biomolecules ∆ntarget = 1.2 ×10−8 RIU. The GH signal ~18 µm was enhanced by six orders of magnitude compared to those of pure silver ones (1.1027 × 10−5 µm). Thus then, sensitivity with the atomically thin perovskite metasurfaces is 1.2862 × 109 µm/RIU, which is more than 106 times than 918.9167 µm/RIU for silver sensing substrate with the same thickness. To realize the ultimate goal of single molecular and even submolecular detection, we have also used the effective medium theory (EMT) from Equations (11) and (12), the Lumerical Finite-difference time-domain method (FDTD) for integrating the 2D perovskite into a plasmonic metasurface structure and obtained a much enhanced GH-SPR sensitivity and also localized electric field enhancement. By optimizing the width from 10 to 60 nm of the silver groove structure for a fixed period of 150 nm, the resonance curve could be further damped and more close to the zero-reflection. The depth of the groove is fixed at 3 nm, which is optimized by the FDTD calculation from 0 to 5 nm . The atomically thin perovskite nanomaterials with high absorption rates enable the precise tuning of the depth in the plasmonic resonance dip. We have also provided our comparison plasmonic results for experimental and simulation data for different layers of atomically thin nanomaterials in 9 µm/RIU. More importantly, a strong localized electric field enhancement was excited between the groove edge. We believe that this enhanced electric field together with the enhanced GH shift would enable the single molecular or even submolecular detection for hard-to-identify chemical and biological markers, including single nucleotide mismatch in the DNA sequence, toxic heavy metal ions and tumor necrosis factor-α (TNFα).An ultrasensitive plasmonic biosensor has been designed in this paper by engineering the atomically thin 2D perovskite nanostructures on the silver metasurfaces. A record-high GH-SPR sensitivity of 1.2862 × 10