The repair of previously scratched monolayers of various adherent tumor cell lines  was inhibited more significantly by the “original” VSL#3 when compared to the “newfound” VSL#3. Tumor cell cycle profile, in particular cell cycle arrest and apoptotic death of the cancer cells, further confirms that the “original” VSL#3 has a better functional profile than the “newfound” VSL#3, at least in in vitro. Our data stress the importance of the production conditions for the “newfound” VSL#3 considering that this product is intended to be used for the dietary management of patients with very serious diseases, such as chronic inflammatory bowel diseases.A careful selection of the probiotic agent, standardization of the dose and detailed characterization of the beneficial effects are essential when considering use of a probiotic for the dietary management of serious diseases. However, changes in the manufacturing processes, equipment or facilities can result in differences in the product itself due to the live nature of probiotics. The need to reconfirm safety and/or efficacy for any probiotic product made at a different factory is therefore mandatory. Recently, under the brand VSL#3®, a formulation produced by a manufacturer different from the previous one, has been commercialized in some European countries (the UK and Holland). VSL#3 is a high concentration multi-strain preparation which has been recognized by the main Gastroenterology Associations for the dietary management of pouchitis as well as ulcerative colitis. We have compared the “original” VSL#3 produced in USA with the “newfound” VSL#3 produced in Italy. According to our results, the “newfound” VSL#3 has 130–150% more “dead bacteria” compared to the “original” product, raising concerns for the well-known association between dead microbes with adverse effects. The abilities of bacterial lysates from the two formulations to influence  This dehe host'  ii) the To make any medical food claim, probiotics are required to demonstrate a beneficial effect through appropriate human clinical trials in the target patient group . Howeverth Triennial Yale/Harvard Workshop on Probiotic Recommendations recognized for the first time level B and C evidence for this probiotic preparation in the management of non-alcoholic fatty liver disease (NAFLD) in adults and children, and in alcoholic liver diseases [For the majority of the marketed probiotics, the available evidence deals mainly with their safety and ability to colonize the gut, rather than adequate clinical efficacy. On the contrary, for the VSL#3 formulation, a large body of clinical evidence has been reported for several groups of patients –10. Thisdiseases . Manufacin vitro on tumor cell lines’ viability, proliferation, cell cycle profile and apoptotic death levels would be significantly different.Recently, under the brand VSL#3®, a formulation produced by a manufacturer different from the previous one, has been commercialized in the UK and Holland, hereafter defined as “newfound” formulation. Our hypothesis was that even though the two formulations are claimed to be the same in terms of species identity and CFU counting, the biological effects of “original” and “newfound” VSL#3 when compared bifidobacteria , four strains of lactobacilli , and one strain of Streptococcus salivarius subspecies thermophilus DSM 24731. Some boxes of VSL#3® distributed by Ferring Pharmaceuticals were purchased in the UK  and also in Holland  for testing and comparison with the product distributed by Ferring in Italy . The Italian product we analyzed was produced in the USA, while the NL and UK products were manufactured in Italy.VSL#3 is a composite probiotic mixture containing multiple strains of three viable lyophilized bacteria species: three strains of Streptococcus thermophilus DSM 24731, bifidobacteria , lactobacilli , while the composition of the “newfound” VSL#3 formulation is reported on the package as: Streptococcus thermophilus BT01, bifidobacteria , lactobacilli   .®13 green fluorescent nucleic acid stain  and the red fluorescent nucleic acid stain, Propidium Iodide (PI) . These stains differ both in their spectral characteristics and in their ability to penetrate healthy bacterial cells. When used alone, the SYTO®13 stain labels bacteria with both intact and damaged membranes. In contrast, PI penetrates only bacteria with damaged membranes, competing with the SYTO®13 stain for nucleic acid binding sites when both dyes are present. When mixed in correct proportions, SYTO®13 stain and PI produce green fluorescent staining of bacteria with intact cell membranes and red fluorescent staining of bacteria with damaged membranes. The background remains virtually non-fluorescent. The standard LIVE/DEAD viability assay  uses SYTO®9 instead of SYTO®13, in this case SYTO®13 is used as an alternative since SYTO®13 proved to have less background in the analysis. 100 mg VSL#3 product was dissolved in 1 ml H2O. 2 μl of the VSL#3 mix was added to 100 μl of staining solution containing 10 μM PI and 8 μM SYTO®13 and stored in the dark at room temperature for 15 minutes. After the incubation the solution was diluted in 1 ml H2O and applied on a glass slide. BacLight™ mounting oil  was applied, and cells were inspected visually using an Olympus BX60 epifluorescence microscope using a FITC or Cy3-specific filter. Live cells stain green and dead cells stain red. Different amounts of cells were applied on the glass slide and 10 microscope views were analyzed.The live/dead status of bacteria in the VSL#3 product was assessed using a mixture of SYTOin vitro experiments, cell lines were treated for different times as below specified, depending on the type of experiment, with the bacterial lysates’ suspensions at 10, 50 and 100 μl/ml , corresponding, respectively to 1, 5 and 10 mg (lyophilised bacteria weight)/ml. According to the manufacturer’s informations, the amounts of bacterial lysates added to the cell cultures corresponded, respectively, to 108, 5x108 and 109 CFU/ml culture medium.Stocks of 1 g of each VSL#3 lot were suspended in 10 ml of PBS. After three freeze-thaw cycles, samples were sonicated  with a Vibracell sonicator . Bacterial cells disruption was verified by measuring the absorbance of sample at 590 nm before and after every sonication step. The sonication steps were repeated until the optical density reached a constant value. Protein concentration was measured using Bio-Rad Dc Protein Assay . The total protein level was comparable in all bacterial lysates’ samples (range 5–6 mg protein/ml). For the xg. Jurkat cells were plated 5x105 cells for each condition. HT1080 cells were cultured in DMEM supplemented with 10% (v/v) of FCS and with 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin in a monolayer culture. HT1080 cells were seeded into a sterile tissue culture 12-well plate  at 18,000 cells/cm2 for each experimental condition. Caco-2 cells were maintained in culture in DMEM supplemented with 10% FCS, 1% non-essential amino acid, 1 mM sodium pyruvate and 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin and seeded at 18,000 cells/cm2 for each experimental points. Under these culture conditions, this cell line remained proliferative and undifferentiated. For adherent cells, after reaching 80% confluence adherent cell cultures were expanded using a trypsin solution from bovine pancreas (Sigma-Aldrich). The well plates were incubated in sterile conditions at 37°C in a 5% CO2 humidified atmosphere. The complete medium was totally replaced every three days. All culture reagents were acquired from Euro Clone .Acute human T leukemia-derived Jurkat cell line and human fibrosarcoma HT1080 cell line were acquired from ATCC ; human colon adenocarcinoma-derived Caco-2 cell line were purchased from Sigma-Aldrich . Jurkat cells were routinely grown in plastic culture flasks containing RPMI 1640 medium supplemented with 2% FCS, 2 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 μg/ml). Subculturing was performed every 2–3 days and the culture medium was totally replaced by centrifugation for 10 min at 400xg and the pellets incubated with a 0.04% trypan blue solution for 5 min. Afterwards, cells were transferred to a Bürker counting chamber and counted by microscopy . Dead cells looked totally blue stained and living cells appeared normally bright and colourless. The cell number counts as well as the percentage of live and dead cells were thus registered. Non-treated cells were also analyzed and served as negative controls.Cultured cells were collected at 12, 24 and 48 hours and live cells were counted after trypan blue dye  exclusion. Cells were incubated with bacterial lysates at 1, 5 or 10 mg/ml, as above described, for 12–48 h, after which were washed with PBS, centrifuged for 10 min at 400in vitro monolayer wound healing assay, cell cultures were allowed to proliferate until ~90% confluence was reached, then DMEM was removed from the well and cell monolayers were scratched using a 200 μl pipet tip to create a uniform cell-free wound area with reproducible width of wounding (0.7 mm width). Debris was removed from the culture by gently washing with sterile PBS. Cell cultures were incubated with fresh medium at 37°C in a 5% CO2 humidified atmosphere in the presence or absence of bacterial lysates at 1, 5 or 10 mg/ml, as above described. Non-treated cells were also analyzed and served as negative controls.As previously described , for theCell migration was monitored by microscopy using an inverted light microscope  and photographed from time 0 (T0), after 15 hours (T15h), and 22–24 hours. The last time point to observe the total scratched monolayer closure after the injury in the untreated cells was in the range 24–28 hours. The images acquired for each sample were further analyzed quantitatively as illustrated below. The amount of wound closure by cell migration consisting in calculation of percentage of closure of the wound was performed using TScratch, a software tool developed to automatically analyze wound healing assays. It is available as a stand-alone application using Matlab .xg for 10 min at 4°C, counted and fixed adding of cooled ethanol solution at 70% in PBS with gentle mixing at 4°C for 30 min. Fixed cells (106 cells/ml) were transferred to plastic BD tubes , washed twice with ice-cold PBS and stained with solution containing 50 μg/ml Propidium Iodide, 0.1% Nonidet-P40 and RNase A (6μg/106 cell) for 30 min in the dark at 4°C. Cell cycle phase-distribution was analyzed by a flow cytometry system. Data from 10,000 events per sample were collected and analyzed using FACS Calibur (Becton Dickinson) instrument equipped with cell cycle analysis software (Modfit LT for Mac V3.0). Apoptotic cells were also determined by their hypochromic subdiploid staining profiles and analyzed using Cell Quest software (BD Biosciences). Non-treated cells were also analyzed and served as negative controls.For cell cycle and apoptosis analysis, Jurkat, HT1080 and Caco-2 cell lines were treated for 24 or 48 h with 10 mg/ml bacterial lysate. After treatment, the cells were collected, washed with PBS, collected by centrifugation at 400®13  staining was also used to reveal apoptotic cell death level, as previously described [®13 and directly used for flow cytometry. Analysis was carried out using Cell Quest software (BD Biosciences).In addition, the detection of nucleic acid-containing microparticles generated in vitro from cell lines through SYTOescribed . Treatedxg. Cytosolic fractions were placed in duplicate into wells of streptavidin-coated microplate, to which was added the immunoreagent containing a mixture of anti–histone-biotin and anti–DNA-POD. The plate was incubated for 2 hours at room temperature on a MP shaker at 300 rpm. During the incubation period, the anti-histone antibody binds to the histone component of the nucleosomes and simultaneously captures the immunocomplex to the streptavidin-coated microplate via its biotinylation. At the same time, the anti–DNA-POD antibody reacts with the DNA component of the nucleosomes. The unbound antibodies were washed with incubation buffer. The amount of nucleosomes retained by the POD in the immunocomplex, corresponding to the extent of apoptosis was determined photometrically with ABTS  as substrate using microplate reader at a wavelength 405 nm and reference wavelength of 490 nm. Obtained data were expressed as Arbitrary Unit/106 cells.DNA fragmentation was measured by quantitation of cytosolic oligonucleosome-bound DNA, using an enzyme-linked immunosorbent assay (ELISA) kit , according to the manufacturer's instructions. This assay determines apoptosis by measuring mono- and oligonucleosomes in the lysates of apoptotic cells. Briefly, after treatment, the cells were collected and washed with PBS, then the cytosolic fraction was extracted by centrifugation at 200P<0.05 was considered statistically significant.Data (mean ± SD) were analyzed using Prism 5.0 GraphPad Software, San Diego, Ca. For comparison between two means, Student’s unpaired t test was used. For comparison of groups, a one or two-way ANOVA test followed by Bonferroni post hoc test were used. The boxes containing the product were handled according to the manufacturer’s instructions. The box purchased in Italy  contained the probiotic mix produced in the USA, a formulation already utilized by our group for previous publications in 2001, 2008 and 2011 and other unpublished tests –18  the number of dead bacteria present in the product is not disclosed.P<0,0001) and a markedly higher percentage of dead cells (P<0.0001) when compared to the “original” VSL#3 formulation (P<0.0001). These differences can be clearly visualized from the microscopy images  the capability of the product to colonize the gut properly is related to the number of “live” bacteria per sachet; (b) the quantity of “dead” bacteria is a good predictor of the quality of the product and its stability, and (c) “dead” bacteria are not an inert material. Both lots of “newfound” product contained a considerably lower percentage of live cells , each sachet of “original” VSL#3 will contain 242 billion dead bacteria  cell lines. The effect of bacterial lysates at 1, 5, and 10 mg/ml on the rate of closure of scratched monolayers was analyzed and compared to the relative untreated cells, as described in Methods’ section. The wound closure (percentage of closure), in either untreated or bacterial lysate-treated cells, was evaluated by observing in microscopy the re-populating area between the wound margins at different time intervals after the lesion. In order to quantitatively analyze the effects of bacterial lysates on wounded area resurfacing, the images obtained by inverted light microscope proliferation were acquired and converted to % closure by using a mathematical system able to automatically calculate the portion of area occupied by the cells. In In order to further compare the ability of “original” and “newfound” VSL#3 to influence cell proliferation and migration, an in vitro biological effects of “original” and “newfound” VSL#3 bacterial lysates, their ability to influence cell cycle distribution and apoptosis level on tumor cell lines was also assessed. In To further comparatively investigate the Tumor cells appeared to be differently affected by treatment with bacterial lysate samples. Cytofluorimetric analysis indicated that a G0/G1 cell cycle arrest and a parallel G2/M percentage reduction were induced in Jurkat cells by addition of “original” VSL#3 lysate. This effect was statistically significant either when compared to untreated cells or to “newfound” VSL#3 sample. On the other hand, the “newfound” VSL#3 formulation didn’t significantly influence the Jurkat cell cycle distribution.The treatment with “original” VSL#3 bacterial lysate of both HT1080 and Caco-2 tumor cells led to a significant increase of the S phase cells with a parallel reduction of both G0/G1 and G2/M phase %. The results were statistically significant when compared either to untreated cells and “newfound” VSL#3-treated cells. The effect of “newfound” VSL#3 formulation even if able to influence with a similar trend the cell cycle distribution, was definitely much weaker as compared to that obtained with the “original” product. Indeed, a statistically significant difference was observed between “original” and “newfound” VSL#3 effects on cell cycle distribution either on HT1080 or Caco-2 cells.The results listed in In order to examine whether “original” and “newfound” VSL#3 bacterial lysates can promote apoptotic death in our tumor cell systems, cell death was monitored by cytofluorimetry, as described in the Methods section. In our experimental conditions, Jurkat, HT1080, and Caco-2 tumor cells appeared to be all influenced, even if at different levels, by treatment for 48 h with “original” and “newfound” VSL#3 bacterial lysates at 10 mg/ml. “Original” VSL#3 product resulted more efficient in inducing a statistically significant increase of apoptosis level in all analyzed tumor cell lines when compared either to untreated cells or “newfound” VSL#3 bacterial lysate. VSL#3, a multi-species probiotic mixture, is widely used clinically, mainly to treat mild to moderately active inflammatory bowel disease . It alsoare often defined by their manufacturing processes. Changes in the manufacturing process, equipment or facilities could result in changes in the biological product itself and sometimes require additional clinical studies to demonstrate the product's safety, identity, purity and potency.” Sanders et al. [Bifidobacterium animalis subsp. lactis INL1 [4 logs higher than for the cells grown at pH 5.0. Additionally, further analysis by electron microscope found that the culture grown at pH 6.5 showed production of extracellular compounds (exopolysaccharide type) not found in the same strain grown at pH 5.0. Authors found that these extracellular compounds could exert an in vivo response not found with the strains grown at lower pH. pH sensitivity of a given probiotic strain appears to be not only influenced by the pH level in culture production but also by the type of cryoprotectant used for freeze-drying of strains. Saarela et al (2009) [Lactobacillus rhamnosus was variable depending not only on the fermentation pH level but also on the use of either polydextrose vs sucrose as a cryoprotectant. Kimoto-Nira et al. (2008) [Bifidobacterium lactis dependent on the type of fermentation broth used to grow the strain. Specifically, the interleukin-12, a cytokine which played a key role in the gut mucosal immune system response, was found to be enhanced in cell lines by B. lactis when grown on M17 fermentation media vs MRS media. In addition, the strains were found to have different cell wall compositions, at the sugar and fatty acid levels. Both the cell wall and immunomodulatory differences indicate that “sibling” strains of B. lactis grown using different technologies will yield bacteria with different clinical functionality.While the “original” VSL#3 formulation is not regulated as a drug, the guidance of the FDA with respect to biologic products is nevertheless relevant. As the FDA notes repeatedly in numerous guidance documents: “In contrast to chemically synthesized small molecular weight drugs, which have a well-defined structure and can be thoroughly characterized, biological products are generally derived from living material-human, animal, or microorganism- are complex in structure, and thus are usually not fully characterized” . The FDAs et al.  have anatis INL1  was growl (2009)  showed t. (2008)  reportedLactobacillus rhamnosus GG, may differ depending on the product and source of the strain, thus concluding that the original properties used in the selection of specific probiotic strains may be influenced by industrial production processes and, consequently, influence the outcome of human intervention studies. The examples above mentioned merely hint at the variables that can impact the functionality of a given probiotic. It is the reason why FDA requires for biosimilar “generic” biologics drugs comparative human clinical data in addition to in vitro comparative assays. In the case of the “original” VSL#3 formulation the complexity is further compounded by the fact that the product contains eight strains, which are all individually cultivated and lyophilized. Variations in their properties may affect the results of experimental studies as well clinical outcome of therapy interventions. In the present study, “original” and “newfound” VSL#3 formulations, both of them distributed under the “VSL#3” brand name in the UK, Holland and in Italy by the same company, were comparatively analyzed both for the bacterial composition and their ability to influence in vitro tumor cell lines. The results indicate significant differences between the two formulations in these properties under the same laboratory conditions. In our hands, the two formulations contained a significant different number of live and dead cells and differently influenced tumor cell viability, proliferation, cell cycle distribution and apoptosis level. Live viable probiotics seem to have superior efficacy to dead bacteria, both in vivo and in vitro, according to the literature [Grześkowiak et al (2011)  have repterature  and gastterature .in vitro. According to our results obtained by comparing the “newfound” VSL#3 to the “original” VSL#3, this is not the case here.Any changes brought to probiotic formulations and/or probiotic manufacturing processes should not be considered relevant and the product should be considered substantially equivalent, if they do not (a) compromise any original probiotic properties, (b) do not reduce the delivery of metabolically active, live bacteria, and, (c) do not change the effects of the product on epithelial and immune cell functions


Topological crystalline insulators are merely protected by individual crystal symmetries and exist for an even number of Dirac cones. Here, we demonstrate that Bi-doping of Pbx1−SnxSe (111) epilayers induces a quantum phase transition from a topological crystalline insulator to a Z2 topological insulator. This occurs because Bi-doping lifts the fourfold valley degeneracy and induces a gap at Topological insulators constitute a new phase of matter protected by symmetries. Time-reversal symmetry protects strong topological insulators of the Z 2 topological insulator by doping Bi into Pbx1-SnxSe (111) thin films.Transitions between topological phases of matter protected by different symmetries remain rare. Here, Mandal et al. report a quantum phase transition from a topological crystalline insulator to a Z Provided that the system stays in the same symmetry class of the Hamiltonian2, it is fundamentally impossible to follow a path from this topologically distinct phase of matter to a trivial phase without closing the insulating bulk band gap. For a strong topological insulator, metallic surface states are necessarily present at the boundary to a trivial insulator as, for example, air or vacuum. These topological surface states are protected by time-reversal symmetry and their energy vs. momentum dispersion mimics quasirelativistic, massless particles, with the shape of a Dirac cone and a peculiar helical spin texture4. The topological classification is given by the so-called Z2 invariant ν0, which for odd number of Dirac cones is ν0 = 1, giving rise to strong (Z2) topological insulators, but for even number of cones is zero, characterizing weak topological or trivial insulators6.Topological insulators are bulk insulators with a metallic surface state2) topological insulator to a trivial insulator by alloying as has been shown for Bi2Se3:In8 and BiTl(Sx1−Sex)29. The fundamental principle of bulk-boundary correspondence dictates again that this topological phase transition proceeds through a transition point where the bulk band gap closes. In this picture, it is supposed that the crystal symmetry is maintained through the phase transition. However, the crystal symmetry itself can protect topologically distinct phases as well, termed topological crystalline insulators (TCIs)14. For TCIs, the decisive role of the crystal symmetry renders the topological protection dependent on the specific crystal face10. The topological invariants allow for an even number of Dirac cones, which are, however, not robust against disorder5. Pbx1−SnxSe and Pbx1−SnxTe represent such mirror-symmetry protected TCIs with fourfold valley degeneracy11 in which the trivial-to-TCI phase transition is reached for sufficiently large Sn contents15. Upon cooling, the lattice contracts and the enhanced orbital overlap leads to an inverted  bulk band gap, which, via bulk-boundary correspondence, gives rise to Dirac cone surface states. This has impressively been shown by temperature-dependent angle-resolved photoemission (ARPES)15.It is possible to transform a strong  surface can be gapped22. In that case, the topological phase remains, however, unchanged by the symmetry breaking and the Z2 invariant stays even. It is crucial for the present work to note that in principle the topological phase does not need to be preserved by a distortion. Indeed, topological phase transitions have recently been predicted for two-dimensional TlSe23 and three-dimensional SnTe by distortions24 and by finite-size effects25.On the other hand, there are crystals that are not necessarily topological, but change their symmetry and their electronic properties with temperature. Such phase-change materials have been studied intensively for non-volatile data storage because their properties can be altered dramatically at the structural phase transitionx1−SnxSe by cooling through the complete trivial to topological phase transition. We demonstrate a new type of phase transition from crystal-symmetry-protected to time-reversal symmetry-protected topology controlled by Bi incorporation. We show that when Bi is introduced in the bulk making the system n-type, a gap is opened up at the one Dirac cone at the center of the surface Brillouin zone at , while the three Dirac cones located at the x1−SnxSe, that is, are gapless at low temperature. Our findings provide the first experimental evidence for a topological phase transition from a TCI with an even number of Dirac cones to a Z2 time-reversal symmetry-protected strong topological insulator where the number is odd (three). Following the recent prediction24, the origin of the novel topological phase transition is interpreted as due to a sublattice shift and rhombohedral distortion along the [111] direction, which lifts the bulk band inversion only at the Z-point (L-point in the undistorted phase) projected onto Here, we investigate the band topology of the (111) surface of Pbx1−SnxSe films of high quality by molecular beam epitaxy  cleavage plane of bulk crystals previously studied26, for the (111) orientation, the four bulk L-points project on the following four time-reversal invariant surface momenta: 27. This is seen in the ARPES data shown in Fig. 28 or by observation of the appearance of the Dirac cones in ARPES26. Indeed, as seen in Fig. xSn = 10%, Fig. xSn = 20%  Pb11 Pb1−xS20% Fig.  and xSn28% Fig. , the gap0.72Sn0.28Se. When substitutionally incorporated at cation  lattice sites, the group V element Bi acts as electron donor due to its excess valence electron29. Increasing the Bi concentration nBi thus leads to a strong upward shift of the Fermi level by 200 meV for nBi = 2.2% , for Bi doping nBi < 0.6%, no difference in the gaps Δ at xSn > 16%), the Dirac cones at x1−SnxSe:Bi as a strong Z2 topological insulator already for moderate Bi doping. This is the central result of the present work. While a TCI is protected by mirror symmetry, the odd numbered Dirac cones of strong topological insulators are protected by time-reversal symmetry and robust against disorder. Figure 2 phase, which has never been observed before for this material class, exists in a wide temperature range from ~150 K down to the lowest temperature probed in our experiments.The effect of Bi on the Dirac cones at the }Γ¯ Fig.  and \doc}M¯ Fig.  close sy}M¯ Fig. , the founts Fig. , but ope0 K Fig. . Thus, wx1−SnxSe26 occurs between trivial  from trivial to Z2 topological by cooling and (ii) from topological crystalline to Z2 topological by adding Bi. The resulting topological phase diagram  is not sufficient to stabilize the cubic bonds35. Thus, SnTe and GeTe assume a rhombohedral structure and even for PbTe minute addition of <0.5% of Ge suffices to drive Pbx1-GexTe into the rhombohedral phase, rendering it ferroelectric37. Thus, very small changes on the group IV lattice sites give rise to structural phase transitions. Indeed, for our Bi-doped films, we find a small Bi-induced rhombohedral distortion along the [111] direction using x-ray diffraction  orientation of our films, this symmetry breaking lifts the even number degeneracy of the Dirac cones such that a gap is opened only at the In general, all IV–VI compounds are close to such a structural phase transition due to their mixed covalent-ionic bonding. They belong to the family of 10 electron systems and crystallize either in the cubic rock salt, rhombohedral, or orthorhombic structure. As calculated by Littlewood33, covalently bonded and crystallizing in a rhombohedral structure. Accordingly, when incorporated at group IV lattice sites in Pbx1−SnxSe, it shifts the alloy toward a more covalently bonded structure in the phase diagram39. Moreover, on such lattice sites, Bi also reduces the cation vacancy concentration and the latter strongly enhances the ferroelectric Curie temperature TC of the cubic-to-rhombohedral phase transition40. Indeed, in SnTe thin films with reduced vacancy concentration, ferroelectricity up to room temperature was recently reported38. Thus, both effects of the Bi, higher covalency, and lower vacancy concentration, contribute to structural symmetry breaking and trigger the novel TCI-to-Z2 topological phase transition discovered in the present work.Finally, we would like to address the role of Bi as decisive ingredient for symmetry breaking even at small concentrations. Bi is another 10 electron systemx1−SnxSe films on BaF2 substrates was performed using molecular beam epitaxy (MBE) in ultrahigh vacuum conditions better than 5 × 10−10 mbar at a substrate temperature of 380 °C. Effusion cells filled with stoichiometric PbSe and SnSe were used as source materials, as well as a ternary Pbx1−SnxSe source with xSn = 25%. Bi-doping was realized using a Bi2Se3 effusion cell. The chemical composition of the layers was varied over a wide range from xSn = 0 to 40% by control of the SnSe/PbSe beam flux ratio, and a two-dimensional growth was observed by in situ reflection high-energy electron diffraction  Pb11 Pb1−xS−10 mbar. ARPES measurements were performed at the UE112-PGM2a beamline of BESSY II at pressures better than 1 × 10−10 mbar using linearly polarized p + s photons incident on the sample under an angle ϕ = 45°. We used photon energies between 18 and 23 eV for the temperature-dependent ARPES measurements of the band dispersions, and a photon energy of 90 eV for the core levels  and 0.3° (angular).For the ARPES measurements, the films were capped in situ in the MBE chamber with a 200 nm thick amorphous Se layer at room temperature to protect the surface against oxidation during transport to the BESSY II synchrotron radiation source in Berlin, Germany. There the Se cap was completely desorbed in the ARPES preparation chamber by annealing at about 230 °C for 15 min in 3 × 10The composition of the epilayers was determined using high-resolution x-ray diffraction and the Vegard’s law (Supplementary Fig. 4 cm2 V−1 s−1 in dependence of the carrier concentration.The dopant concentration and transport properties were assessed by Hall effect measurements at 77 K (Supplementary Fig. The authors declare that all data supporting the findings of this study are available within the paper and its Supplementary Information


N-acylpyrazole substrates. All reactive intermediates remain bound to the catalysts thereby providing a robust catalytic scheme (no exclusion of air necessary) with excellent stereocontrol. This work expands the scope of stereocontrolled bond-forming reactions of photoexcited intermediates by providing catalytic asymmetric access to a key nitrogen heterocycle in organic chemistry.The reactivity of photoexcited molecules has been extensively studied for decades but until today direct bond-forming reactions of such excited states in a catalytic and asymmetric fashion are restricted to the synthesis of cyclobutanes via [2 + 2] photocycloadditions. Herein, we demonstrate a previously elusive visible-light-induced catalytic asymmetric [2 + 3] photocycloaddition of alkenes with vinyl azides. A wide range of complex 1-pyrrolines are obtained as single diastereoisomers and with up to >99% enantiomeric excess using a simple reaction setup and mild reaction conditions. The reaction is proposed to proceed through the photoexcitation of a complex out of chiral rhodium catalyst coordinated to α,β-unsaturated  Despite intensive research on photoexcited molecules, stereocontrol of direct bond formation upon photoexcitation remains limited. Here the authors expand the research on stereocontrolled bond forming photochemistry and introduce the catalytic asymmetric synthesis of chiral nitrogen heterocycles. Typically, photochemically excited species engage in single electron transfer with a substrate or reagent to generate radical ions and/or free radicals, which then participate in a multitude of reaction schemes 30 developed in our group  (Table 1) of the pyrazole, the yield of 1-pyrroline 3c was increased to 83% with 87% ee (entry 3). Optimization of solvent and concentration led to an improved outcome of 3c with 98% yield and 95% ee in CDCl3 under more diluted conditions (entries 3–6). We continued to evaluate the effect of the auxiliary in order to reduce the catalyst loading . Importantly, this simple and mild protocol can be conducted under air without the need for inert conditions, which renders it very practical (entry 12). It is noteworthy that the iridium analog Λ–IrS31 also provided the 1-pyrroline 3f (50% yield) albeit without any enantioselectivity (entry 13). Control experiments demonstrated that the current transformation relies both on rhodium catalyst (entry 14) and visible light (entry 15).Recently, we introduced a visible-light-activated intermolecular [2 + 2] photocycloaddition of photoexcited substrate-bound rhodium-based Lewis acid complexes with alkenes providing a practical synthetic route to enantioenriched cyclobutanes1 to the chiral rhodium catalyst generates the substrate/catalyst complex I, which upon visible-light excitation generates the excited state I*, most likely in its triplet excited state. The following direct reaction of the excited state I* with the vinyl azide cosubstrate 2 affords the catalyst-bound diradical intermediate II under generation of a new C–C bond, with the stereochemistry of this bond formation being controlled by the metal-centred chirality of the rhodium complex. Subsequent extrusion of dinitrogen delivers the catalyst-bound iminyl radical intermediate III32, which then undergoes a stereoselective cyclization generating the product coordinated complex IV. Release of product from the rhodium catalyst and coordination of new substrate 1 then initiates a new catalytic cycle. Since the reaction is insensitive to air, it can be assumed that the diradical intermediates II and III are very short-lived and thus cannot engage in any side reactions, thus revealing the advantages of such direct bond-forming reactions from a photoexcited state over processes that occur through initial electron transfer.The proposed mechanism for this 2 + 3] photocycloaddition is shown in Fig.  + 3 phot1f bound to the (racemic) catalyst RhS shown in Fig. RhS–1f) confirms the proposed formation of intermediate I of the catalytic cycle. Interestingly, it further reveals a π–π stacking between the cyclometalating benzothiazole moiety of the rhodium catalyst and the p-methoxyphenyl moiety of the pyrazole auxiliary, which might explain the superiority of this auxiliary by modulating binding constant, steric and/or electronic effects33. Second, UV-Vis absorption spectra demonstrate that the visible-light absorption of substrate 1f is greatly enhanced upon binding the rhodium catalyst (RhS–1f) so that in the reaction mixture the intermediate I is selectively photoexcited to generate the photoexcited intermediate I* from which the reaction (I* + 2 → II) occurs  or position  of the substituents. Heteroaryl frameworks were proved to be compatible as demonstrated by the effective formation of thienyl- and indolyl- substituted 1-pyrrolines . Notably, this protocol is amenable to the construction of complex 1-pyrrolines bearing a quaternary stereocenter (3q–s), including a spiro center (3s), reflecting the robustness of this single rhodium catalysis. Additionally, α,β-unsaturated N-acylpyrazoles (E)-1q and (Z)-1q gave the product 3q in similar yields with identical stereochemistry as an indicator for the involvement of the diradical intermediates II/III. To our delight, various functionalized vinyl azides were readily accommodated, including not only aryl vinyl azides (3t–x) but also more challenging alkyl vinyl azides (3z–ab). Although vinyl azides bearing an electron-rich substitution at phenyl group gave a decreased ee value , the reaction outcome was not influenced by steric hindrance (3v–x). Furthermore, a free hydroxyl group (3aa) as well as C = C double bonds (3ab) were tolerated, thereby highlighting the good functional group compatibility of the current protocol.With the mechanistic picture in mind, we next evaluated the generality of the [2 + 3] photocycloaddition with a single chiral rhodium catalyst using 3-(4-methoxyphenyl)pyrazole as the auxiliary. As shown in Fig. 6 could be obtained by the [2 + 3] photocycloaddition with ethisterone derived vinyl azide 5 in 86% yield with almost a single stereoisomer pyrazole was fully recovered in these conversions. Furthermore, 1-pyrrolines with a prochiral cyclic imine group is applicable for further stereoselective synthetic manipulation as highlighted by the synthesis of a carbapenem analog 8 and pyrrolidine 10.With the practical conditions and broad substrate scope in hand, we next evaluated the synthetic potential of this protocol Fig. . We weremer Fig. . Besidesmer Fig. . Althoug34 access to the highly relevant class of chiral 1-pyrrolines based on a visible-light-activated [2 + 3] photocycloaddition of alkenes with vinyl azides catalyzed by a chiral rhodium complex. The employed robust rhodium-based chiral Lewis acid catalyst is key to the successful implementation of this methodology. Upon binding to the alkene substrate, it provides a unique handle for selective visible-light excitation of the substrate/catalyst complex with the triplet spin being mainly localized at the alkene carbons so that an efficient reaction with the vinyl azide cosubstrate can occur, while the metal-centered chirality provides outstanding stereocontrol. In addition to the conceptual appeal, the synthetic usefulness of this protocol is indicated by the simple reaction setup with a single catalyst and no necessity for inert reaction conditions, exceptional stereoselectivity with virtual perfect diastereoselectivity and up to >99% ee, a broad substrate scope that gives access to a large variety of chiral 1-pyrrolines, including quaternary stereocenters, and the ability to use these 1-pyrrolines as building blocks for conversions into biologically relevant chiral pyrrolidines. We anticipate that this report will stimulate the further discovery of new types of stereocontrolled reactions with electronically excited molecules.In summary, we here introduced an important expansion of catalytic asymmetric reactions occurring via direct stereocontrolled bond-forming reactions of photoexcited states Fig. . Previou1H and 13C NMR spectra of the compounds in this article, see Supplementary Figs 8 and 10, see Supplementary Figs For the emission spectrum of the blue LEDs lamp and the exemplary reaction setup, see Supplementary Figs N-acylpyrazole 1g , Λ–RhS , CDCl3 (1.0 mL) and vinyl azide 2a  were added in sequence under open air conditions. Then, the tube was sealed and positioned at ~8 cm from a 24 W blue LEDs lamp. After stirred at room temperature for 24 h (monitored by TLC), the mixture was directly subjected to 1H NMR to determine the diastereoselectivity of 3g as only a single diastereoisomer formed. Then, all the mixture was collected and purified by flash chromatography on silica gel (n-hexane/EtOAc) to afford the product 3g as a white solid . The enantiomeric excess of 3g was determined as 99% ee by HPLC using a Chiralpak OD-H column (mobile phase = n-hexane/isopropanol 95:5).Exemplary, to an oven-dried 10 mL Schlenk tube with a stirring bar, α,β-unsaturated RhS–1f and 3k reported in this article have been deposited at the Cambridge Crystallographic Data Centre (CCDC) under deposition numbers CCDC 1568748 and 1568749, respectively. The data can be obtained free of charge from The Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif. The authors declare that the data supporting the findings of this study are available within the article and its Supplementary Information Files. All other data are available from the authors upon reasonable request.The X-ray crystallographic coordinates for the structure of Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1


Experiential learning is crucial for the development of all learners. Literature exploring how and where experiential learning happens in the modern clinical learning environment is sparse. We created a novel, web-based educational tool called “Learning Moment” (LM) to foster experiential learning among our learners. We used data captured by LM as a research database to determine where learning experiences were occuring within our emergency department (ED). We hypothesized that these moments would occur more frequently at the physician workstations as opposed to the bedside.We implemented LM at a single ED’s medical student clerkship. The platform captured demographic data including the student’s intended specialty and year of training as well as “learning moments,” defined as logs of learner self-selected learning experiences that included the clinical “pearl,” clinical scenario, and location where the “learning moment” occurred. We presented data using descriptive statistics with frequencies and percentages. Locations of learning experiences were stratified by specialty and training level.A total of 323 “learning moments” were logged by 42 registered medical students  and 13 MS 3 over a six-month period. Over half (52.4%) intended to enter the field of emergency medicine (EM). Of these “learning moments,” 266 included optional location data. The most frequently reported location was patient rooms . Physician workstations hosted the second most frequent “learning moments” . EM-bound students reported 43.7% of “learning moments” happening in patient rooms, followed by workstations (32.8%). On the other hand, non EM-bound students reported that 66.3% of “learning moments” occurred in patient rooms and only 8.4% at workstations (p<0.001).LM was implemented within our ED as an innovative, web-based tool to fulfill and optimize the experiential learning cycle for our learners. In our environment, patient rooms represented the most frequent location of “learning moments,” followed by physician workstations. EM-bound students were considerably more likely to document “learning moments” occurring at the workstation and less likely in patient rooms than their non EM-bound colleagues. Experiential learning is critical for successful growth and the development of new skills and behaviors. Kolb’s four-part experiential learning model, which incorporates concrete experience, reflective observation, abstract conceptualization, and active experimentation, can be used in a recurring cycle that supports progressive new learning .1 While The classic model of bedside teaching has been in decline.What do we already know about this issue?Despite playing a crucial role in learner development, there is little literature exploring how and where experiential learning is happening in the modern clinical learning environment.What was the research question?We used data from “Learning Moment” (LM) to determine where learning experiences were occuring in our emergency department (ED).What was the major finding of the study?Patient rooms represented the most frequent location of “learning moments” in our ED, followed by physician workstations.How does this improve population health?Using LM to determine the location of learning experiences has the potential to inform the design of optimal learning ecosystems and maximize experiential learning for future trainees.Learning Moment” (LM) that integrates the principles of asynchronous learningWe created a novel, web-based educational tool called “Our goal in this study was to use data captured by LM as a research database to determine where learning experiences were occuring within our emergency department (ED) clinical learning environment. We hypothesized – based on our own experiences and on recent literature (described above) demonstrating that faculty-learner interactions often occur in locations away from the bedsidewww.learningmoment.org ) to encourage the reflective and abstract conceptualization steps of Kolb’s experience learning model. The LM platform  tertiary care hospital that hosts a postgraduate year 1–4 EM residency with 12 residents per year, as well as robust third- and fourth-year medical student (M3 and M4) clerkships. As part of the monthly student orientation, MS3s and MS4s were introduced to and registered on the LM platform by research assistants. Demographic data, including the student’s intended specialty and year of training were collected at the time of user registration. Students were encouraged to use our platform to log learning experiences during their clerkship without a formal requirement to do so. Students logged self-selected learning experiences in the form of “learning moments” that included the clinical “pearl,” clinical scenario, and location where the “learning moment” occurred, among other optional data variables. Students were encouraged to log onto LM and view the Community Feed to read about “learning moments” logged by their peers.Learning Moment Reflection” sessions led by experienced clinical faculty in small-group format. These sessions provided medical students with additional opportunities to discuss and expand upon the “learning moments” that they had already logged in order to deepen their understanding through further reflection and abstract conceptualization. A link to the LM website was made accessible directly from the electronic medical record to promote ease of access. Our study was approved as exempt by our institutional review board.While peer reflection can promote critical thinking, previous work has stressed the role of faculty facilitators to provide oversight in the reflective process.We presented data using descriptive statistics with frequencies and percentages, and we stratified locations of learning experiences by specialty and training level. Fisher’s exact test was used to compare the distribution of locations between groups. We used SAS v9.3  for all data analysis.Of the 53 medical students who completed their EM clerkship rotation, 42 (79.2%) logged at least one “learning moment.” A total of 323 “learning moments” were logged between August 22, 2016 – February 12, 2017, spanning the course of six one-month-long clerkship rotations. The MS group consisted of 29 MS4s and 13 MS3s. Over half (52.4%) intended to enter the field of EM while the remainder were either undecided or intended to train in another specialty .The median number of “learning moments” logged by these students was six (interquartile ratio=7.5). Nearly 40% (n=16) of the students logged 1–4 “learning moments,” and over 25% (n=11) logged 5–8 “learning moments” .A total of 266 “learning moments” included optional location data. The most frequently reported location was patient rooms . Physician workstations hosted the second most frequent “learning moments”  .The distribution of reported locations of “learning moments” differed between EM-bound and non EM-bound students. EM-bound students reported 43.7% of “learning moments” happening in patient rooms, followed by workstations (32.8%), hallways (14.8), and resuscitation rooms (8.2%). On the other hand, non EM-bound reported 66.3% of “learning moments” having occurred in patient rooms and only 8.4% at workstations (p<0.001).Differences were also seen in the distribution of “learning moment” location between MS3s and MS4s. MS3s logged 41 (68.3%) “learning moments” happening in patient rooms with the remainder evenly divided between workstations, hallways, and resuscitation rooms. MS4s logged relatively fewer, 94 (45.6%), “learning moments” happening in patient rooms and more, 61 (29.6%), happening at the workstations (p=0.005).LM is a novel educational platform designed to integrate experiential learning and shared learning. We employed the LM platform to track the location of “learning moments” as a proxy to gain insight into the location of experiential learning in a busy ED. Our data suggest that the majority of “learning moments” occur at the patient bedside, despite our hypothesis to the contrary. By hypothesizing that the majority of “learning moments” on LM would occur away from the bedside, where faculty-learner interactions have been shown to be low,The decline of bedside teaching and the pressures and barriers influencing it have been well-described.The results of this study indicate that the physician workstation is the most common non-bedside location of “learning moments,” especially for EM-bound students. It is likely of benefit to optimize educational experiences in this setting as well. Multiple potential strategies to teach effectively at workstations have been described in the literature.Interestingly, EM-bound students reported notably fewer “learning moments” occurring in patient rooms and more occurring at the workstations than their non EM-bound colleagues. These findings raise the question: Are there inherent differences between the learning preferences of EM-bound students vs. non EM-bound students? Considering that aversion to bedside rounding is commonplace among EM residents, one could postulate that those who choose EM as their intended specialty may have an increased propensity to learn “on the spot,” such as at the workstations where presentations of cases commonly occur. It is conceivable that EM-bound students may preferentially receive more teaching from residents and faculty who are aware of their learners’ decision to enter EM. The shift in location of “learning moments” from bedside to workstations from the MS3 to MS4 year may represent changing learning preferences as students progress along the spectrum from novice to expert learners, or may simply be the result of a large proportion of rotating MS4s who chose EM as their intended specialty compared to mostly undecided MS3s . It woulOur study has several limitations. We emphasized during orientation and registration to LM that the “location” drop-down menu when logging a “learning moment” on the website was meant to indicate the location of where the “learning moment” occurred, not the location of the patient. Students may still have mistakenly chosen the patient’s room number when they logged some “learning moments.” Doing so would have falsely elevated the number of “learning moments” documented in patient rooms. Furthermore, out of 323 total “learning moments”, only 266 contained the optional location data. Our study is limited by the use of learner self-selected learning experiences in the form of “learning moments” as an imperfect proxy to gain insight to the location of experiential learning in the ED environment. No direct observation of where learning was happening was performed. Recall bias is a significant confounder to our data as we intentionally left it up to the students to choose what learning experiences to log as a “learning moment.” Certain experiences may be overlooked when learners fail to consciously notice that learning is happening. They may also choose to document certain types of learning experiences over others. Lastly, our pilot was conducted at a single department within one institution. Other learning environments may yield different results. Nevertheless, data from LM has the potential to help educators better understand the intricacies of local learning microenvironments as well as the broader clinical learning ecosystem.LM was implemented within our ED as an innovative, web-based tool to fulfill and optimize the experiential learning cycle for our learners. In our environment, patient rooms represented the most frequent location of “learning moments,” followed by physician workstations. EM-bound students were considerably more likely to document “learning moments” occurring at the workstation and less likely in patient rooms than their non EM-bound colleagues. Although successfully piloted in the ED, LM is potentially adaptable to other clinical departments and institutions as we seek to inform the design of optimal learning ecosystems and maximize experiential learning for all future trainees. Efforts are ongoing to make LM available to more learner populations in new learning environments as we continue to demonstrate the feasibility and value of our platform to various stakeholders throughout health professions education.


Intracerebroventricular injection of AMP also suggested possible involvement of central activation of AR signaling. AMP, induced HMS in a strain and age independent fashion and did not affect the behavioural and reproductive capacities. AMP induced HMS, mitigated radiation-induced oxidative DNA damage and loss of HSPCs. The increase in IL-6 and IL-10 levels and a shift towards anti-inflammatory milieu during the first 3–4 hours seems to be responsible for the augmented survival of HSPCs. The syngeneic bone marrow transplantation (BMT) studies further supported the role of radiation-induced inflammation in loss of bone marrow cellularity after TBI. We also showed that the clinically plausible mild hypothermia effectively mitigates TBI induced lethality in mice.Therapeutic hypothermia has proven benefits in critical care of a number of diseased states, where inflammation and oxidative stress are the key players. Here, we report that adenosine monophosphate (AMP) triggered hypometabolic state (HMS), 1–3 hours after lethal total body irradiation (TBI) for a duration of 6 hours, rescue mice from radiation-induced lethality and this effect is mediated by the persistent hypothermia. Studies with caffeine and  Non-hibernating mammals can barely survive a few tens of minutes without oxygen or reduced Tc4. The extreme ability of hibernators to survive otherwise lethal conditions generated significant interest, for obvious implications, in emergency medicine5. In 2008, Blackstone et al. have demonstrated that hydrogen sulfide (H2S), a small gaseous molecule, can trigger a deep HMS in laboratory mouse a naturally non-hibernating animal which later on showed to be protective against a variety of stressors10. However, H2S failed to render similar effect in larger naturally non-hibernating mammals thereby raising concerns about its translational success11. In fact the requirement for higher concentrations and the inability of H2S to inhibit the mitochondrial complex IV due to small window of opportunity has been proposed to be some of the reasons for the failure in large mammals12. Nevertheless, amongst different molecules which have been identified to be effective HMS inducers, adenosine monophosphate (AMP) can be considered important. Initially in 2006, Zhang et al. have identified 5′-AMP, a natural metabolite and an approved neutraceutical, as a key mediator of torpor like state in mice13. Since then AMP has been intensively investigated as a pharmacological means for induction of hibernation like state in natural non-hibernators and is known to protect model organisms from a number of shocks including oxidative stress16. Interestingly, the synthetic torpor has also been proposed to have implications in the management of cancer17. During HMS, tissues tolerate higher radiation doses and it has direct implications in radiotherapy of cancers as higher doses of radiation can be targeted to tumors which normally kill an awake and non-hibernating cancer patient. Apart from cancer patients, HMS has also been proposed to be a promising tool for deep space travel. As it ensures low metabolic consumption, it may lead to reduced requirement for food, liquid and life support systems which have direct consequences on payload which in turn critically determines long space flights19. However, practical application of HMS in deep space travel is still a hypothetical area and may take decades before it can be realized. Nevertheless, synthetically induced HMS can have significant immediate medical applications, including radiation protection17.Hibernating animals survive extreme environmental settings, including anoxia, freezing temperatures by suppressing their metabolic rate (<5% of resting rate), reducing core body temperature  triggered HMS modulate radiation induced early inflammatory response and rescued both long- and short-term hematopoietic stem cells (HSCs) in the bone marrow leading to effective hematopoietic recovery and increased survival.a) and relative humidity of 23–25 °C and 55% respectively. Unlimited mouse chow  and tap water was provided ad libitum, and the animals were maintained on a 12 hours light and 12 hours dark cycle. All the efforts were made to minimize animal number and suffering. All experiments were performed following the protocols approved by the Committee on the Ethics of Animal Experiments of INMAS, Delhi, India.Animal handling and experiments with mice were carried out in accordance with the approval from the Committee on the Ethics of Animal Experiments, Institute of Nuclear Medicine and Allied Sciences (INMAS), Defence Research and Development Organization (DRDO), Delhi, India . Male and female mice  of 8–10 weeks of age (25 ± 2 g), were used for this study. For some experiments, 8–10 weeks old female Sprague Dawley rats weighing 180 ± 10 g were used. The animals were kept at Experimental Animal Facility, INMAS, Delhi, India at an ambient temperature . For radio-mitigation studies, mice were exposed to 8Gy (C57BL/6J hereafter referred to as B6) or 7Gy (BALB/c) at a dose rate of 1.00Gy/minute on a rotating chamber and the irradiation field size was 35 × 35 cm2. Animals were partially restrained and then exposed to irradiation in a group of 4. All irradiations were consistently carried out between 12:00 to 01:00 p.m. to minimize the chronosensitivity33. The mapping of exposure rates was carried out by an Atomic Energy Regulatory Board (AERB) authorized health physicist using a calibrated RadCal 0.6 cm3 therapy grade ion chamber and electrometer system. One hour after TBI, mice were either treated with vehicle  or AMP  via intraperitoneal route (i.p.). In some instances, amifostine 34 was administered i.p., 8 hours after TBI and AMP treatment. For evaluating the radiomitigative effect of adenosine A1 receptor (A1AR) agonist,6N-cyclohexyladenosine 35. Animal’s overall health, appearance, body weight and mortality were recorded daily till the end of the experimentation (till 30 post-irradiation days) and the survival was compared over the first 30 days between each of the treated groups and the radiation-only control group. The overall health of non-irradiated mice which were administered with either AMP (0.5 mg/g b.w.) or CHA (1 mg/kg b.w.) was free from adverse side effects over the period of study (30 days). After different treatments, animals were housed and maintained at experimental animal facility without any supportive care. Animals showing a combined score of more than 8  were humanely euthanized to minimize the pain and distress36.TBI was performed using a et al.37. HMS was induced by administering 0.5 mg/g b.w. of AMP  through i.p. route. The animals were then kept at room temperature (25 °C) or at a Ta of 15 °C (for maintaining HMS) for a total duration of 6 hours before they were brought back to room temperature for recovery. The mice which did not respond to the treatment and were awake during the incubation at 15 °C were removed from the study. To evaluate the role of AR in AMP mediated HMS, caffeine (40 mg/kg b.w.) was administered (i.p.) 30 minutes prior to the administration of AMP. Detailed methodology for measurement of Tc, surface temperature (Ts), peripheral blood flow, activity profile, tissue ATP levels, blood glucose, pyruvate and lactate levels, hif1-α expression, haematological profiles, and spatial dynamics of blood components during the course of HMS is presented in supplementary information. Bone marrow cell count, cytologic evaluation, HSPCs enumeration, alkaline comet assay, cytokine measurements in serum and syngeneic BMT was also done to assess the effect of HMS on radiation induced hematopoietic form of acute radiation syndrome (hARS) (for detailed methodology see supplementary information).HMS was induced using AMP by essentially following the approach reported by Daniels b measurements were done (for detailed methodology see supplementary information).For establishing the role of central activation of the AR in induction of hypometabolic state by AMP, direct intracerebroventricular (i.c.v) injection of AMP and TAll the results are presented as mean ± standard error of the mean (SEM). All statistical analysis were performed with GraphPad Prism from GraphPad Software . Two way ANOVA repeated measure followed by boneferoni post-test was used for multiple comparisons and Student’s t-test was used for single comparison. The kinetics of overall survival, defined as time from the date of irradiation to the date of death for the mice under study, was analysed using log-rank test (two-tailed). Differences with a p-value less than 0.05 were considered statistically significant.All relevant data are contained within the article and Supplementary files, or are available from the authors on request.c regardless of whether Ta was 25 or 15 °C  displayed a decrease in Tc within 15 minutes which gradually reduced to a minimum value (~30 °C) reached after 1 hour. Thereafter, the Tc then gradually increased and reached to normal levels by 3 hours. However, mice that were treated with AMP (+Ta 15 °C) exhibited a steep decrease in Tc and by 2 hours the Tc plummeted to near Ta (~16 °C). The Tc remained at near ambient temperature until the animals were shifted from Ta of 15 °C to Ta of 25 °C and within 2 hours of being at 25 °C the Tc returned to normal levels (~36 °C). In about 1 hour after the administration of AMP (+Ta 15 °C) the animals were in a state of deep sleep without any movement except occasional involuntary movements (Supplementary video). Unlike AMP (+Ta 15 °C) treated animals, the untreated mice placed at a Ta of 15 °C were found to be active except that they were cuddling, to minimize the heat loss. Mice exposed to TBI and treated with AMP also responded in a similar fashion and the reduction and recovery kinetics of Tc was found to be similar to that of un-irradiated control mice treated with AMP (+Ta 15 °C)  treatment almost completely abolished the induction of HMS. In caffeine treated animals after 1 hour of AMP administration the Tc reached to a minimum (~28 °C) level which thereafter recovered to normal levels by 3 hours. Similarly, AMP mediated HMS lead to induction of hypoxia in both brain  Fig. . To veriain Fig.  and femuain Fig.  which wa40. Downstream activators of oxidative stress and electromagnetic pulse are known to regulate the expression of AR42. A key question raised from these studies was whether AMP can induce reversible HMS in lethally irradiated mice akin to normal un-irradiated mice. Mice exposed to TBI and treated with AMP exhibited a reduction and recovery kinetics of Tc similar to that of un-irradiated control mice treated with AMP (+Ta 15 °C) and found to be mediated through AR signalling  induced 1 hour after TBI (hereafter referred to as TBI + HMS group) resulted in a significant increase in the survival advantage (~60%) over vehicle treated TBI (+Ta15 °C) (hereafter referred to as TBI group) by day 30 . The radiation doses selected for this study, 8Gy is known to effectively induce hARS and yield around 85–90% thereby enabling survival of a few animals (15%) which is critically required for monitoring the changes in irradiation alone group and for comparing the mitigative effect of irradiated animals treated with AMP. B6 male mice exposed to TBI (8Gy) followed by placing them at Tays Fig. . In cont 30 Fig. . Adminis 30 Fig. . Althoug 30 Fig. .Figure 2a, TBI caused a massive loss of WBC (>88%), lymphocytes (90%) and granulocytes (87%) 24 hours after TBI when compared to vehicle treated un-irradiated (+Ta15 °C) control group (hereafter referred to as control), . AF, which was administered 8 hours after induction of HMS in TBI animals also failed to prevent the radiation induced loss of WBC but significantly enhanced their reclamation on day 30 (70% recovery) when compared to TBI (p = 0.001) or TBI + HMS groups (p = 0.001). Correspondingly, both the lymphocytes and granulocytes, have shown a response and recovery similar to that of WBCs , Fig. . On day Evaluation of histopathological changes, on day 30, in longitudinally sectioned and H&E (Haemotoxylin and Eosin) stained femurs showed that, TBI caused aplasia with few islands of nucleated hematopoietic cells and massive deposition of adipose cells Fig. . HMS sig−Sca1−c-kit+ cells) in vehicle treated animals after 24 hours. HMS, although statistically not significant when compared to TBI group, apparently countered radiation induced depletion of HPCs and the surviving fraction was found to be 24%  and 30%  after 24 hours , a fraction of BM which contains the HSCs, in vehicle treated animals within 24 hours to 17% , and LT-HSCs (Lin−Sca1+c-Kit1+CD34− cells) were found to be surviving after 24 hours, in TBI animals. HMS rescued both ST-HSCs  and LT-HSCs  from radiation induced depletion. AF reinforced the radiomitigative effect of HMS and the surviving ST-HSCs and LT-HSCs were found to be 72% (p = 0.011) and 66% (p = 0.033) respectively  , 42 (p = 0.003), 47(p = 0.009) and 38% (p = 0.033) for HPCs, KSL, ST-HSCs and LT-HSCs respectively which further lowered to 22 (p = 0.031), 22 (p = 0.0001), 43 (p = 0.004) and 24%; (p = 0.0004) after 24 hours. However, HMS ameliorated TBI induced changes in MMP and a higher number of KSL and ST-HSCs with Rh123high status were observed both at 6 hours  and 24 h . In case of HPCs, HMS influenced the MMP status in a complex manner and at 6 hours 79% of progenitors were found to with high MMP which quickly declined to 24% after 24 hours. Interestingly, HMS did not prevent radiation induced loss of MMP in LT-HSCs at 6 hours (39% vs 38% in case of TBI group) but after 24 hours more number of LT-HSCs (44%) with high MMP were present as compared to TBI animals (24%). Administration of AF further enhanced the protective effect of HMS and rescued KSL (54%), ST-HSCs (63%) and LT-HSCs (39%) from radiation induced loss of MMP. Similar to HMS alone, HMS + AF did not prevent radiation induced loss of MMP in HPCs. As radiation induced genomic damage and instability compromises both the short and long term repopulation ability of different subclasses of HSPCs45, the DNA damage in the surviving HSPCs was evaluated. In view of recent evidences suggesting that the observed gamma H2AX foci, a marker for assessing DNA damage, in HSCs may mark cellular events distinct from DNA damage47 we sought to quantify the DNA damage directly in Lin− bone marrow mononuclear cells (BMMNCs) using alkaline comet assay. TBI significantly increased Olive Tail moment (OTM), a reliable end point for DNA damage, when compared to background damage in control  Fig. . TBI sigrol Fig.  after 6 als Fig. . Corroboals Fig. . Within als Fig. .Figure 449. To assess the influence of TBI and HMS, the expression of c-Kit in annexin V− and annexin V+ subsets of different HSPCs was monitored   . Interestingly, irrespective of treatment annexin V+ HSPCs have exhibited c-Kit high status  Fig. . HMS couup) Fig. . AF furttus Fig. .51 and their ratio (IL-6/TNF-α) is indicative of prevailing inflammatory status. TBI elevated the pro-inflammatory status and the IL-6/TNF-α ratio was found to be significantly lower than the control group after 3 hours (p = 0.0093) , MCP-1 , IL12p70 ] while it did not affect the levels of anti-inflammatory cytokines evaluated in this study [IL-6  and IL-10 ] after 3 hours Fig. . HMS sigc reduced to ~32 °C is in clinical practice for the management of cardiac arrest patients52. As reduction of Tc below 30 °C in patients is associated with increased risk, the radiomitigative effect of clinically relevant reduction in Tc (30–32 °C) was tested using CHA, which was previously reported to induce clinically relevant hypothermic state in rodents53  was reached after 2 hours which persisted for the duration of the experiment (6 hours)  Fig. . As was ted Fig. . CHA , 24 hours after TBI, collected from untreated animals effectively protected all the animals from radiation induced lethality. Transplantation of bone marrow grafts  harvested 2 hours after TBI (8.5 Gy) resulted in 65% of animals surviving the duration of the experiment (30 days). While animals which received bone marrow grafts harvested 6 hours after TBI did not show any significant improvement in survival when compared to vehicle treated irradiated groups.The results from this study suggests that pro-inflammatory environment peaks 3 hours after TBI and significantly subsides by 24 hours. HMS exerted the mitigative action by preserving the bone marrow HSPCs from TBI induced inflammatory milieu. To test this in fact is the case and also due to obvious implications in autologous BMT, bone marrow aspirated soon after TBI (2 or 6 hours) was tested for its ability to rescue recipients from lethal TBI (8.5 Gy) Fig. . All aniet al. (2010) have shown using genetically modified mouse models that the modulation of purine nucleotide pool in erythrocytes and increased production of 2,3 bisphospho glycerate and allosteric modulation of oxygen binding to haemoglobin plays an important role in induction of hypometabolic state37. However, central activation of adenosine receptor following AMP administration has also been proposed by other investigators40. Consistent with earlier reports, intraperitoneal pre-administration of caffeine abrogated the effect of AMP, suggesting possible involvement of central activation of adenosine receptor in AMP induced HMS . This difference could be attributed to possible involvement of impaired oxygen delivery due to changes in glycolysis in erythrocytes apart from central activation of adenosine receptors in case of i.p. administration which might have resulted in deeper reduction in Tc. The dose used for i.c.v. administration was more than what was used in earlier reports40, suggesting that AMP dose may not be a factor contributing to reduced Tc observed in case of animal which received AMP into i.c.v. region.The results demonstrate that irradiated animals respond to AMP triggered HMS akin to normal counterparts which is critical for its utility in translating to radiation exigencies. Previously, Daneils HMS Fig. . However54 which raises concerns about its utility in managing radiation over exposure victims. In fact, infection has shown to be a common complication of patients undergoing therapeutic hypothermia55. However, lack of any disease symptoms in mice which underwent HMS suggests that transient disappearance and resting immune status during a 6 hours HMS may not have adverse effects, suggesting that the approach may have practical utility. It could be attributed to the shorter duration for which the animals were in HMS unlike hibernators or during therapeutic hypothermia where the duration is much longer.The disappearance of different subsets of WBCs from peripheral blood during HMS is akin to a situation of immune compromised state58. Hypoxic milieu restricts the quality and quantity of free radicals generated upon irradiation and has been suggested to be the mechanism of action for a number of radioprotectors including AMP56. The present study suggests that apart from the persistent HMS, the reduction in generation of secondary free radicals due to the induction of hypoxia seems to be contributing to the overall radiomitigative effect. Elevated expression of HIF-1α, a reliable marker of hypoxia, both in brain  and in femur (bone marrow) supports the notion. In fact it is well known that HMS induced during hibernation or after induction of synthetic torpor leads to induction of tissue hypoxia. The inability of the transient reduction in Tc, in AMP treated animals placed at 25 °C, to mitigate radiation induced lethality supports the notion that persistent HMS is critical but nevertheless HMS mediated tissue hypoxia seems to be a significant contributory factor.AMP is known to protect mice when administered 15–60 min prior to lethal TBI and induction of hypoxia and enhanced post-irradiation repair have been suggested to be the responsible mechanismsc reducing to ~32 °C, to mitigate radiation induced lethality suggests that persistent HMS is a critical determinant rather than the depth. This is in line with the beneficial effect of clinically practiced therapeutic hypothermia52. Apart from the duration, the timing of induction also seems to be important as induction of HMS later than 3 hours after TBI did not mitigate radiation induced lethality (data not shown). This suggests that initial 3–4 hours are critical for the success of HMS as radiomitigative strategy and is in line with earlier observations that the timing determines success of a given mitigative strategy59. Antioxidants are effective mostly when administered prior to irradiation, repair enhancing agents effective when administered shortly after irradiation and cell proliferation inducing agents are effective when administered from 24 hours to few days after irradiation59. The results also suggests that HMS modulated early events which contribute to the loss of HSCs. Among different deleterious events contributing to the loss of HSCs during early post-irradiation scenario, the elevated levels of inflammogens, loco regional inflammation and protracted oxidative stress seems to be the key players62. We supported this by showing that HMS shifts the pro- and ant-inflammatory balance towards later by increasing the expression of IL-6 and IL-10 leading to the rescue of different HSPCs in bone marrow. This is in line with earlier reports that systemic hypothermia elevates IL-6 levels65. However, enhanced proliferation of surviving stem cells contributing to the increased number of HSPCs observed after 24 hours in TBI + HMS group cannot be ruled out. As AMP mediated AR signalling, IL-6 and IL-10 are known to regulate and expand HSPCs67.The ability of moderate hypothermia, with T68. The better hematopoietic recovery observed in animals which have received AF 8 hours after induction of HMS could be attributed to the free radical scavenging and cytoprotective effects of AF69 which probably effectively countered the protracted oxidative stress. CD117 (c-Kit) which marks different HSPCs has also been implicated in regulation of haematopoiesis71. The inability of lethally irradiated mice to recover from the depressed hematopoietic system could be attributed to the significant increase in the expression of c-Kit. This assumption is in line with earlier studies, with gain and loss-of-function c-Kit mutants, suggesting that subtle changes in c-Kit signalling profoundly impacts HSCs function73. More recently, Shin et al. have shown that c-Kithigh HSCs selectively undergo apoptosis in response to 5-FU exposure34. The present study also demonstrated that annexin V+ cells in different subsets of HSPCs are highly c-Kit positive. Interestingly, the SEM for MFI of annexin V+ fraction was found to be much higher than for corresponding HSPCs in annexin V− fraction indicating that annexin V− cells are more homogeneous in terms of c-Kit expression when compared to apoptotic fraction. It is interesting to evaluate whether cells at different stages of the apoptotic cascade exhibits differences in the expression of surface c-Kit. It seems that HMS delayed the entry of different subsets of HSPCs (except HPCs) into proliferation phase post-irradiation thereby preventing them from being exposed to ensuing inflammogens and protracted oxidative stress resulting in increased survival. This assumption is in line with the observation that HMS prevented radiation induced increased expression of c-Kit and largely retained different subsets of HSPCs in c-Kit low status. Similarly, the loss of different subsets of HSPCs in case of irradiated animals could be attributed to the increased entry into division phase, soon after irradiation, which in fact is known to result in their loss68.HSCs, post irradiation, are known to exit dormancy and enter into proliferation phaseet al. who reported that elevated TNF-α levels in inflamed bone marrow contribute to compromised reconstitution of donor bone marrow74. We supported this by demonstrating that transplantation of bone marrow, collected soon after lethal TBI but prior to initiation of radiation induced inflammatory wave (within 3 hours), can rescue lethally irradiated mice. The inability of the bone marrow collected 6 hours after lethal irradiation to protect mice from radiation could be attributed to irreversible loss due to ensuing inflammatory milieu in bone marrow. However, mobilization of surviving HSPCs to extra-medullary hematopoietic sites75 leading to reduced number of critical HSPCs in bone marrow collected 6 hours after TBI cannot be ruled out. The countermeasures which can mitigate radiation effects when administered at least 24 hours later are expected to be relevant for mass radiation exposures and the requirement for induction of HMS soon after TBI (within 3 hours) is a critical hurdle in the practical utility of the current approach. However, considering the well accepted clinical practice of therapeutic hypothermia, the immediate translational advantage in using AMP or other small molecules seem to be promising which warrants further investigations in higher mammals.In this study AMP was found to be effective when administered, 1–3 hours post-irradiation which indicates that sufficient number of undamaged and functionally competent quiescent HSCs survive even after lethal irradiation which with time succumb to ensuing inflammatory wave. This is in line with a recent report by Ishida Video 1Dataset-1