Raphanus raphanistrum) and hybrid (R. raphanistrum x R. sativus) populations using a life table response experiment.As global climate change transforms average temperature and rainfall, species distributions may meet, increasing the potential for hybridization and altering individual fitness and population growth. Altered rainfall specifically may shift the strength and direction of selection, also manipulating population trajectories. Here, we investigated the role of interspecific hybridization and selection imposed by rainfall on the evolution of weedy life-history in non-hybrid (r) and asymptotic (λ) population growth rates and sensitivities, a measure of selection imposed on demographic rates. Hybrid populations experienced 8.7-10.3 times stronger selection than wild populations for increased seedling survival. Whereas crop populations generally exhibit little dormancy and wild populations often exhibit dormancy, non-hybrid populations experienced 10% stronger selection than hybrid populations for exhibiting seed dormancy. Selection on survival-to-flowering in wild, not hybrid, populations declined marginally with increasing soil moisture. Hybrid populations exhibited greater r, but not λ, than wild populations regardless of moisture environment. In general, fecundity contributed most to differences in λ but fecundity only contributed positively to hybrid λ relative to wild λ when precipitation was altered (either higher or lower than control) and not under control watering conditions.In documenting long-term population dynamics, we determined intrinsic (Selection on key demographic traits may not change dramatically in response to rainfall, and hybridization may more strongly influence the demography of these weedy species than rainfall. If hybrid populations can respond to selection for increased dormancy, this may make it more difficult to deplete weed seed banks and increase the persistence of crop genes in weed populations. Hybridization between crops and their wild and weedy relatives instantaneously changes the genetic composition of weed populations  and thusIf fitness advantages of advanced-generation hybrids are context-dependent, then one might predict that environmental gradients should be important influences on population demography . For insProviding that abiotic selection pressures vary across environmental clines , 22, theGeographic and temporally shifting climatic conditions can affect plant phenology and fecundity, key fitness-related traits . In partLactuca and Raphanus, or above- and below-ground biomass of Brasica rapa and B. napus hybrids , these λ in the crop-wild Raphanus complex ).ca. seven days at 30 °C. For 30 randomly selected, reproductive plants per sub-plot , we counted the number of fruits per plant. Number of seeds per fruit was assessed by counting locules in a silique for 10 randomly chosen fruits per plant. To estimate the number of seeds per plant, we multiplied the average number of seeds per fruit by the number of fruits.Plants were harvested as they senesced or at the last weekly census, after the first frost when there was no new fruit development . Above-ground biomass was collected in paper bags and dried in an oven for λ) of each experimental population each year  as a linear function of biotype (g), watering treatment (w), and their interaction (gw): − λgw = λ(⋅⋅) + αg + βw + αβgw where αg is the effect of the gth level of the biotype, βw is the effect of the wth level of the watering treatment, and αβgw is the interaction of the gth biotype and wth watering treatment, measured relative to the projected growth rate of a reference matrix (..).We used a fixed-effect life table response experiment  to compTo obtain the treatment matrices, we first averaged all replicates of matrices belonging to a given treatment combination  , 55. We δλ/δaij) from the relationship δλ/δaij = viwj/<w,v > where aij is a matrix element in the ith row and jth column, and v and w are the right and left eigenvectors of the matrix and evaluated the sensitivities, halfway between the reference and treatment matrices ).ronments  and RaphUltimately, studies of crop-wild hybridization assess whether crop-derived traits will persist in wild or weedy populations , 76. OneRaphanus and to ultimately develop predictions about the direction and pace of evolution under changing moisture conditions. Although field studies of selection in genetically diverse experimental populations growing under natural and manipulated conditions are transforming our understanding of selection dynamics [Raphanus populations.Our goal was to develop a quantitative understanding of the selection pressures experienced by weedy dynamics , 82, fewdynamics , 56–58.


Alginate is a commonly used bioink in 3D bioprinting. Matrix stiffness is a key determinant of mesenchymal stem cell (MSC) differentiation, suggesting that modulation of alginate bioink mechanical properties represents a promising strategy to spatially regulate MSC fate within bioprinted tissues. In this study, we define a printability window for alginate of differing molecular weight (MW) by systematically varying the ratio of alginate to ionic crosslinker within the bioink. We demonstrate that the MW of such alginate bioinks, as well as the choice of ionic crosslinker, can be tuned to control the mechanical properties  of 3D printed constructs. These same factors are also shown to influence growth factor release from the bioinks. We next explored if spatially modulating the stiffness of 3D bioprinted hydrogels could be used to direct MSC fate inside printed tissues. Using the same alginate and crosslinker, but varying the crosslinking ratio, it is possible to bioprint constructs with spatially varying mechanical microenvironments. Moreover, these spatially varying microenvironments were found to have a significant effect on the fate of MSCs within the alginate bioinks, with stiffer regions of the bioprinted construct preferentially supporting osteogenesis over adipogenesis. One way of overcoming this limitation is through the use of emerging additive biomanufacturing strategies2. In particular, 3D bioprinting4 allows for the development of complex anatomically accurate scaffold geometries that also mimic aspects of the composition and organisation of native tissues through the simultaneous deposition of biomaterials, cells, proteins and/or genes in defined locations6.Tissue engineering (TE) is a promising strategy for replacing, repairing or regenerating damaged tissues and organs. TE strategies typically incorporate cells, biomaterials and signals (e.g. growth factors), with the goal of developing a construct that once implanted will promote tissue regeneration. A limitation of current TE strategies is their relatively poor spatial control of the distribution of cells, matrix components and bioactive cues within the engineered construct9. It is a highly biocompatible hydrogel whose physical properties can potentially be tailored to direct 3D cell growth and differentiation both in vitro and in vivo12. Moreover, alginate is characterised by a wide pore size distribution (5–200 mm) which facilitates the diffusion of large molecules in and out of the gel13. Alginate is also commonly used for drug/growth factor delivery, where the degradation rate of the alginate can be tuned by altering the MW of the alginate15, which can in turn vary the release rate of the drug/growth factor encapsulated. Gelation of alginate is typically induced by cations such as Ca+, making it an attractive choice as a bioink for 3D bioprinting20. At present the most frequent choice of ionic crosslinker is Calcium Chloride (CaCl2) as it typically leads to rapid gelation due to its high solubility in aqueous solutions21. Calcium sulphate (CaSO4) and calcium carbonate (CaCO3) are the other two popular choices of crosslinkers, however due to their lower solubility within an aqueous solution the gelation time is slower. The choice of ionic crosslinker can also have a significant effect on the mechanical integrity of an alginate hydrogel-based bioink22.One of the main challenges with bioprinting cell laden constructs is the identification of an appropriate bioink, as the material not only needs to have the necessary structural and mechanical properties, but should also protect the cells from damage during printing and ultimately provide them with an appropriate environment to direct or control their phenotype and function. One of the most common natural materials used for hydrogel based tissue engineering and drug delivery is alginatein vivo tissue regeneration is that it is in general non-degradable by mammals, as they lack the enzymes needed to break down the polymer chains21. One application where degradability is vital is in bone tissue engineering, as residual biomaterial can impede vascularization and bone formation26. The degradation rate and mechanical properties of alginate gels can be varied by adjusting its MW29. One of the most common methods of reducing the MW of the alginate is through γ-irradiation. To date, the majority of in vivo bone tissue engineering approaches that utilise alginate as a hydrogel use a γ-irradiated version of this biomaterial as it has significantly faster degradation14, which correlates with increased bone formation within segmental defects34. In the context of bioprinting with alginate, decreasing its MW reduces its viscosity, making it difficult to print with. Therefore, there is likely a trade-off to ensure the MW of an alginate is low enough to degrade in vivo but high enough to have a viscosity suitable for printing.One of the potential limitations associated with the use of alginate for In vivo cells have also been shown to adhere, contract and migrate through an array of tissues varying from soft brain tissue to the stiff osteoid of remodelling bone35. The mechanical properties of the extracellular matrix influence focal adhesion structure and the cytoskeleton of differentiating cells41. Furthermore, the differentiation pathway of MSCs is strongly influenced by the rigidity of the underlying substrate43. MSCs cultured on soft collagen coated gels exhibit spindle-like characteristics of primary neurons, whereas on stiffer matrices MSCs have been shown to adopt a cuboidal osteoblast morphology35. Matrix stiffness has also been shown to regulate MSC differentiation in 3D alginate gels, with adipogenesis supported in soft alginate hydrogels (Young’s Modulus of 2.5-5kPa) and osteogenesis supported in stiffer hydrogels (Young’s Modulus of 11–30 kPa)43. This suggests that by modulating the stiffness of an alginate bioink we might be able to regulate MSC fate within a 3D bioprinted construct.The MW of alginate, choice of crosslinker and gelling conditions have all been shown to have a significant effect on the physical properties of alginate. Furthermore, it has been demonstrated that alginate stiffness is a key determinant of MSC differentiation. It is unclear, however, how these factors impact the printability of alginate, cell viability post-printing and ultimately the phenotype of progenitor cells embedded within 3D bioprinted constructs. The four main objectives of this study were: (1) to investigate the effect of MW, gelling conditions and choice of crosslinker on the bioprintability and mechanical properties of alginate bioinks, (2) to determine how such bioprinting conditions regulate MSC viability post-printing, (3) to examine how the rate of growth factor release from alginate bioinks is influenced by its MW, and (4) to demonstrate that spatially modulating the stiffness of 3D bioprinted constructs can be used to regulate MSC fate and hence engineer complex tissues. We compared the printability, mechanical properties and post-printing cell viability associated with two alginate-based hydrogels of varying molecular weights using a range printing conditions. We then investigated how these parameters can regulate MSC fate within a spatially defined 3D bioprinted construct.4 as a crosslinker resulted in the lowest spreading ratio of 4.3 ± 0.77. At any lower crosslinking ratios, the bioink became over crosslinked and made it very difficult to recapitulate a prescribed printing pattern as seen in Fig. 2 crosslinker that resulted in the lowest spreading ratio of 5.3 ± 0.28, see Fig. To establish the optimum printing parameters, the printability of the two MW alginates  was evaluated at a variety of crosslinking ratios (using bioinks that were prepared in high glucose DMEM). The width of the printed filament was measured using ImageJ software and divided by the internal needle diameter, to obtain the spreading ratio. Lower spreading ratios are desirable to allow the fabrication of cell laden hydrogel structures with high precision. The lowest spreading ratio for the high MW alginate was observed at a crosslinking ratio of 25:9  regardless of the choice of crosslinker, see Fig. 44 that the mechanical properties of alginate depend on phosphate concentrations. Therefore, preparing an alginate solution in PBS over DMEM can significantly affect both the mechanical properties and hence printability of an alginate bioink. It was found that preparing a high MW alginate solution in PBS over DMEM at the same crosslinking ratio (25:9) significantly increased the spreading ratio if CaCO3 was used as the crosslinker, see Supplementary Fig. 2 is used as the crosslinker, see Supplementary Fig. It has been previously established1, as well as any post-printing crosslinking. Therefore, these factors must be considered when determining the viability of the bioprinting process. We first sought to evaluate the chemical cytocompatibility of the three crosslinkers in isolation from other printing factors. To this end, bioinks were prepared exactly as if they would be prepared for printing but were then cast into an agarose mould to create small constructs. All bioinks regardless of the choice of alginate or crosslinker supported high levels of cell viability, with no differences between the groups, see Fig. 4, before printing using a conical needle which has previously been shown to reduce the levels of shear stress cells are exposed to during printing45. There was high cell viability (85.02% ± 5.94) present within all of the constructs 24 hours after printing see Fig. 47 and comparable to that observed in cast hydrogels Fig. During the printing procedure, there are numerous factors that can affect cell viability. These include gelling conditions, fabrication time and the shear stress the cells experience during the crosslinking and printing procedures29. In agreement with this literature, the mechanical properties of the printed constructs increased with increasing alginate MW, see Fig. 4 compared to the same alginate crosslinked with CaCO3 and CaCl2. This change in mechanical properties may be because the CaCl2 has a high solubility in aqueous solutions, which leads to rapid gelation that can reduce construct mechanical properties. Previous studies have shown that a rapid gelation time leads to non-uniform crosslinking, which in turn can negatively impact mechanical properties22. In contrast, CaSO4 and CaCO3 have lower solubility within an aqueous solution, resulting in slower more uniform gelation that can improve the mechanical properties of the resulting hydrogel. A limitation of calcium carbonate is that it is not soluble in water at a neutral pH, therefore to dissociate the Ca2+ ions from the CaCO3 Glucono-δ-lactone needs to be added to lower the pH22. This lowering of the pH may be negatively impacting the mechanical properties of the bioink.As previously stated, the molecular weight of alginate has a significant effect on the mechanical properties of an alginate hydrogel+ ions to cause slower gelling and a reduction of mechanical integrity44. This reduction in mechanical properties was only seen in the high MW alginates crosslinked with CaSO4, otherwise there was little difference in mechanical properties between the bioinks prepared in PBS versus DMEM, see Supplementary Fig. 2, however this is such a low concentration of CaCl2 that it should have negligible effect on the crosslinking potential of the bioink. Moreover, all the groups were prepared in the same DMEM so the differences seen between the groups are not due to the extra presence of CaCl2.The Young’s Modulus of an alginate bioink can also be tuned through varying the crosslinking ratio  alone, with stiffness increasing with relative increases in available crosslinker, see Fig. 14 associated with increased bone formation34. To increase its degradability, previous studies have manipulated the MW weights of alginates, with higher molecular weights associated with slower degradation rates21. As expected, in this study the high MW alginate showed little to no degradation over the 21 days in culture regardless of the choice of crosslinker, see Fig. 4 and CaCl2 degraded significantly faster than those crosslinked with CaCO3. Therefore lowering the pH of the solution can significantly affect both the young’s modulus and degradability of the resulting bioink. Taken together, the choice of ionic crosslinker has a significant effect on the degradability but only when using a low molecular weight alginate bioink.Degradability is another critical mechanical property of a hydrogel, specifically those used for tissue engineering approaches, with for example faster degradation49. Similar results were observed in this study, as straight after printing process only 45–75% of VEGF initially loaded into the alginate was present in the printed construct, see Fig. 49, we found that the MW of the alginate also had a significant effect on the percentage of VEGF present post printing. Lowering the MW of the alginate significantly reduced (p < 0.05) VEGF retention within the bioink post-printing. Interestingly, the alginate MW not only influenced VEGF retention post-printing, but also its release from the bioink over time, see Fig. 50. Future studies using alginate as a growth factor delivery vehicle should investigate the addition of other components such as hydroxyapatite51 or laponite52 as they have been shown to facilitate the adsorption and immobilization of VEGF or the use of gelatin microspheres53 to trap the growth factors within the bioprinted hydrogel and thus slow down its release rate.In general, the release rate of proteins from an alginate gel is relatively rapid due to the high porosity and hydrophilic nature of the gelsin vitro culture; see Fig. 57, and specifically that they preferentially differentiate towards an osteogenic lineage within stiffer alginate hydrogels43. These earlier studies exploring the role of alginate stiffness in regulating MSC differentiation incorporated adhesive ligands into the hydrogel. A potential limitation of this study is that we did not include such cell adhesive ligands into the alginate bioinks, and therefore cells may not be able to initially sense the stiffness of their surrounding environment. Mechano-sensation in these bioinks can potentially be explained by a number of different mechanisms. Shortly after hydrogel encapsulation, MSCs will deposit their own pericellular matrix (PCM) and then sense the stiffness of the hydrogel-PCM composite that surrounds them. Cellular volume changes has also been shown to play a key role in mechano-transduction within hydrogels lacking adhesive ligands58. These mechanisms, either alone or in combination, likely play a key role in the substrate stiffness mediated MSC differentiation observed in the printed constructs. In spite of this, the differences seen in this study might have been enhanced if cell adhesive motifs (such as RGD peptides) were incorporated into the alginate bioink. Future studies will investigate the potential of such bioinks for printing complex tissues.Cylindrical MSC laden constructs were bioprinted with spatially varying mechanical stiffness from the core to the periphery. There was high cell viability (87.025% ± 5.94) present within all of the constructs 24 hours after printing see Fig. The results of these studies demonstrate that by using the same alginate and crosslinker, but varying the crosslinking ratio, it is possible to bioprint constructs with spatially varying mechanical microenvironments. Moreover, by varying the printed microenvironment, MSCs can be directed towards distinct cellular lineages. One biomedical application for this is to begin to direct MSCs towards lineages seen in a long bone organ, where a cortical bone tissue surrounds a complex marrow environment supporting adipocytes and other cell populations. Future studies will investigate the co-deposition of modified alginates alongside mechanically reinforcing polymers such as PCL to engineer composite constructs with bulk mechanical properties compatible with implantation into load bearing environments, but with cell-level mechanical environments engineered to promote defined MSC differentiation pathways.4 produces a significantly stiffer bioink than any other ionic crosslinker investigated here. Furthermore, by using the same alginate and crosslinker, but varying the crosslinking ratio, it is possible to bioprint constructs with spatially varying mechanical microenvironments. The stiffness of these printed cellular environments can be spatially tuned to direct the differentiation of encapsulated MSCs. In conclusion, this study demonstrates how the properties of alginate bioinks can be tuned for optimal printability, controlled growth factor delivery and to spatially direct stem cell fate within bioprinted constructs. In this way, we can use 3D bioprinting to engineer spatially defined microenvironments and corresponding cellular phenotypes seen in complex solid organs such as a long bone.When choosing, an alginate based bioink, the molecular weight of alginate, the gelling conditions and the choice of ionic crosslinker all have a significant effect on printability, mechanical properties and protein release from a 3D printed construct. Specifically, the amount of crosslinker, regardless of the choice of crosslinker, needed to generate a printable bioink is 2.5 times greater when using a low MW compared to high MW alginate. Moreover, if a high MW alginate is used CaSOW = ~75 000 g/mol) and low  molecular weights. The alginate solutions were prepared by dissolving the two alginate powders in either high-glucose Dulbecco’s modified eagle medium  or sterile Phosphate Buffer Solution  to make up a final concentration of 3.5% (w/v). Three different ionic crosslinkers were used to crosslink the alginate bioinks; Calcium Carbonate (CaCO3), Calcium Chloride (CaCl2), and Calcium Sulphate (CaSO4). The 60 mM solutions were prepared by dissolving CaCl2 and CaSO4 (All Sigma Aldrich) within deionised water. For the CaCO3 solution a 2:1 (120 mM: 60 mM) ratio of CaCO3 to Glucono-δ-lactone (All Sigma Aldrich) was prepared in deionised water. The 60 mM solutions were autoclaved prior to crosslinking with the alginate solutions. To form bioinks, these solutions were pre-crosslinked by mixing the alginate solutions with either CaCO3, CaCl2, or CaSO4 at varying volumetric ratios (v/v)  using a dual syringe approach as previously described46. The bioinks could crosslink for 30 minutes prior to printing, see Fig. Two sodium alginate powders of varying molecular weights (MW) were used to generate bioinks of high  was used to evaluate the printability of the generated bioinks. All bioinks were printed using a 25 G needle, at a feed rate of 8 mm/s, fibre spacing of 4 mm, and 0/90° pattern. Other varying parameters for printing of the individual bioinks are described in Table 3, CaCl2 or CaSO4 at the pre-determined optimum cross-linking ratios. Using the 3D bioplotter constructs of Ø 16 mm by 2.5 mm high were printed. The printed constructs were further crosslinked post-printing by placing them in a bath of 60 mM CaCl2 for 1 minute.High and low MW alginate solutions of 3.5% (w/v) were prepared in either PBS or high glucose DMEM, as described in Section 4.1, and pre-crosslinked with 60 mM of either CaCO4 and the bioinks were prepared at varying crosslinking ratios . Cylinders of Ø 16 mm by 2.5 mm high were printed to preform mechanical testing. The printed constructs were further crosslinked post-printing by placing them in a bath of 60 mM CaCl2 for 1 minute.To investigate if varying the crosslinking ratio would influence the mechanical properties of printed constructs, a 3.5% (w/v) bioink of high MW alginate was prepared in high glucose DMEM. This alginate solution was pre-crosslinked with 60 mM CaSO59, see Fig. All mechanical tests were performed using a single column Zwick  with a 5 N load cell as previously described3/cm2. All expansion was conducted in normoxic conditions, expanded in growth medium where the media was changed twice weekly. Cells were used at the end of passage 3.Bone marrow derived mesenchymal stem cells (MSCs) were obtained from the femur of a purchased 4-month old porcine donor from an abattoir (Perma Pig) and carried out in accordance with all relevant guidelines and regulations. The bone marrow was removed from the femoral shaft and washed in growth medium, consisting of high-glucose DMEM  supplemented with 10% fetal bovine serum , 2% penicillin (100 U ml−1) streptomycin (100 μg ml−1), . A homogenous suspension was achieved by triturating with a needle. The solution was centrifuged twice at 650 g for 5 min, each time the supernatant was removed. The cell pellet was triturated and the cell suspension was filtered through a 40 μm cell sieve. Following colony formation, cells were trypsinised, counted and re-plated for a further passage at a density of 5 × 106 cells/mL. The bioink/cell suspension was prepared by resuspending the MSCs in a volume of either PBS or growth medium, depending on the experimental group, to generate a final alginate solution concentration of 3.5% (w/v). These cellular/alginate solutions were homogenously mixed using a dual syringe approach. This final cellular/alginate solution was pre-crosslinked with 60 mM of either CaCO3, CaCl2 or CaSO4 at the pre-determined optimum cross-linking ratios, using the dual syringe approach again. The bioinks were then cast in an agarose block to generate constructs of Ø 6 mm by 6 mm high. These constructs were crosslinked post casting by placing them in a bath of 60 mM of CaCl2 solution for 1 minute, to further replicate the post-printing process. Constructs were cultured in growth medium for 24 hours at normoxic conditions, see Fig. Alginate solutions of 4.4% (w/v) were prepared by dissolving the high and low MW alginate powders in sterile PBS or growth medium, as described above. Each bioink was prepared to have a seeding density of 20 × 103, CaCl2 or CaSO4 at the pre-determined optimum cross-linking ratios, and cast in an agarose block to generate constructs of Ø 6 mm by 6 mm high. Each construct was crosslinked again post-casting in a bath 60 mM CaCl2 for 1 minute. Each construct was cultured in growth medium for 21 days in normoxic conditions. Media from each sample was changed twice weekly. At each time point  samples were washed and snap frozen at −80 °C. Samples were lyophilised by placing the samples in a freeze dryer . Briefly, the samples were initially frozen to −30 °C at a rate of −1 °C/min, this temperature was held for 1 hour before rising to −10 °C at a rate of +1 °C/min. Once at −10 °C, this temperature was maintained under a vacuum of 0.200 mBar for 24 hours before the temperature was increased to +20 °C at a rate of +1 °C/min60. Each sample was then weighed using an analytical balance .To determine the degradation rates over time of the two molecular weight alginates, 3.5% (w/v) alginate solutions of high and low MW were prepared in high glucose DMEM. These alginate solutions were pre-crosslinked with 60 mM of either CaCO49. Using a dual-syringe approach 100ng/mL of Human Vascular Endothelial Cell Growth Factor  was added to the alginate solutions. Once the VEGF is mixed thoroughly the alginate solutions were pre-crosslinked with 60 mM CaSO4 at the pre-determined optimum cross-linking ratios for high and low MW alginate, and printed in an agarose mould to generate constructs of Ø 6 mm by 6 mm high. Each construct was cultured in growth medium for 7 days in normoxic conditions. Media from each sample was changed twice weekly. At each time point  media samples were taken and snap frozen at −80 °C. Samples Printed hydrogels were also taken at Day 0 and Day 7 and snap frozen at −80 °C. An enzyme-linked immunosorbent assay  was used to quantify the levels of VEGF released by the alginates. The alginate samples were depolymerised with 1 mL of citrate buffer  for 15 minutes at 37 °C. The cell culture media and depolymerised alginate samples were analysed at the specific time points detailed above. Assays were carried out per the manufacturer’s protocol (R&D Systems) and analysed on a microplate reader at a wavelength of 450 nm.To establish if the molecular weight of the alginate influences protein release, two 3.5% (w/v) alginate solutions of high and low MW were prepared in high glucose DMEM. Two combinations of high and low MW alginate gels were also prepared in a ratio of 75:25, as previously described6, to create a final concentration of 3.5% (w/v). Two bioinks were prepared by crosslinking the alginate solution with 60 mM CaSO4 at two different crosslinking ratios, a soft bioink (crosslinking ratio of 6:1) and a stiff bioink (crosslinking ratio of 25:9). Constructs were printed to create three experimental groups: (1) Homogenous Soft: where the entire construct (Ø = 20 mm) is printed with the soft bioink; (2) Homogenous Stiff: where the entire construct (Ø = 20 mm) is printed with the stiff bioink; and (3) Gradient construct: where the core (inner Ø = 10 mm) of the construct is printed with the soft bioink and the annulus (outer Ø = 20 mm) is printed with the stiff bioink, see Fig. 2 for 1 minute. Following printing the experimental groups were treated in one of two ways: (1) cultured in Osteo:Adipo medium (as described below) for 7 days or (2) taken for mechanical testing both directly after printing. The constructs that were treated to in vitro culture were cultured for 3 days in a 50:50 Osteogenic:Adipogenic Induction Medium, consisting of growth medium plus dexamethasone (100 nM), ascorbic acid (50 µg/mL), β-glycerolphosphate (10 mM), isobutyl-1-methylxanthine (0.5 mM), indomethacin (200 µM), and insulin (10 µg/mL). After 3 days, medium was changed to a 50:50 Osteogenic:Adipogenic Maintenance Medium, which consisted of growth medium plus dexamethasone (100 nM), ascorbic acid (50 µg/mL), β-glycerolphosphate (10 mM), and insulin  for 24 hours. This cycle was repeated for a 7-day cell culture period.To establish if tuning the bioinks mechanical properties would lead to spatially directing MSC differentiation a 4.4% (w/v) alginate solution was prepared by dissolving high MW alginate in growth medium. As described in section 4.5, porcine MSCs were encapsulated in the alginate solution, at a seeding density of 20 × 10in vitro culture constructs were fixed with 4% paraformaldehyde solution prepared in serum free, phenol free DMEM with 0.1% (w/v) sodium azide (Sigma Aldrich), 0.1% (v/v) Triton-X-100 and 0.1% (v/v) Tween-20, for 30 minutes. The constructs were then washed with 0.1% (v/v) Tween-20 solution and using a 10-mm biopsy the core was separated from the annulus of the constructs. Each section was equilibrated with alkaline staining buffer (pH 8.2) consisting of CaCl2 (100 mM), Tris-Hydrochloric Acid (100 mM), 0.1% (v/v) Tween-20 and Magnesium Chloride (50 mM). In the same staining buffer constructs were incubated in 500 µg/mL Naphthol AS-MX phosphate and 500 µg/mL of Fast Blue BB (All Sigma Aldrich) for 60 minutes. Constructs were then washed again with alkaline staining buffer before being equilibrated back to neutral using DMEM containing CaCl2 (100 mM) and 0.1% (w/v) Tween-20. Constructs were then stained with 0.5% Oil Red O in methanol solution for 90 minutes at room temperature. Samples were fixed again in 4% paraformaldehyde solution, as described above, for 30 minutes.After the 7 days of www.graphpad.com). For all comparisons, the level of significance was p ≤ 0.05.Results are expressed as mean ± standard deviation. Young’s Modulus and Equilibrium Modulus analysis for varying crosslinking ratios was examined using one-way analyses of variance (ANOVA) with the addition of Tukey’s correction for multiple comparisons testing. All other quantitative analyses were examined using two-way ANOVA with the addition of Tukey’s correction for multiple comparisons testing. All analyses were performed using GraphPad (GraphPad Software, La Jolla California USA, Supplementary Figure 1


We decompose the spectra into contributions of electronic state blocking and photo-induced band shifts at a carrier density of 8 × 1020 cm−3. Separate electron and hole relaxation times are observed as a function of hot carrier energies. A first-order electron and hole decay of ∼1 ps suggests a Shockley–Read–Hall recombination mechanism. The simultaneous observation of electrons and holes with extreme ultraviolet transient absorption spectroscopy paves the way for investigating few- to sub-femtosecond dynamics of both holes and electrons in complex semiconductor materials and across junctions.Understanding excited carrier dynamics in semiconductors is crucial for the development of photovoltaics and efficient photonic devices. However, overlapping spectral features in optical pump-probe spectroscopy often render assignments of separate electron and hole carrier dynamics ambiguous. Here, ultrafast electron and hole dynamics in germanium nanocrystalline thin films are directly and simultaneously observed by ultrafast transient absorption spectroscopy in the extreme ultraviolet at the germanium M et al. directly and simultaneously observe ultrafast electron and hole dynamics in germanium thin films.Understanding excited carrier dynamics in semiconductors is central to the continued development of optoelectronic devices. Using extreme ultraviolet transient absorption spectroscopy, Zürch  Investigation of the ultrafast photoexcited electronic response in semiconductors has provided invaluable insights into the carrier dynamics and dielectric properties of many materials. Germanium and its alloys with Si have promise for creating mid-infrared optoelectronics4τ, a broadband pulse in the XUV consisting of an isolated attosecond pulse or a short attosecond pulse train generated by high harmonic generationUltrafast transient absorption spectroscopy in the extreme ultraviolet (XUV) is an important technique for studying electron dynamics at the sub-femtosecond and few-femtosecond timescale6810Germanium is a group IV semiconductor with an indirect bandgap of 0.66 eV. Previous optical experiments observed electron scattering from one part of the band structure to another, that is, intervalley scattering, occurring on a few hundreds of femtosecond timescale131415171819204,5 edge can spectrally resolve signatures of electrons in the CB and holes in the valence band (VB) simultaneously, which enables the time- and energy-resolved tracking of electron and hole kinetics. Carrier-induced and heat-induced band shifts are observed in addition to state filling, synthesizing a complete picture of the photoinduced dynamics. The electron and hole relaxation and recombination times within a single experiment on nanocrystalline germanium thin films are successfully characterized.Here, we address the need for measuring carrier dynamics more comprehensively by using ultrafast transient absorption in the XUV region of the spectrum, with specific application of the method to electron and hole dynamics in photoexcited germanium. A model is established that allows decomposing the measured raw transient absorption data into contributions of SB, shifts of the excited state spectrum and broadening. We show how the carrier dynamics can be extracted by separating spin-orbit components in the XUV. Following this, XUV transient absorption at the germanium MAmeas=Ap−A(E) is defined by the difference between the absorbance of the excited (pumped) thin film Ap and the static absorbance A. Depending on the time delay τ between the VIS-NIR excitation (pump) pulse and XUV probe pulse, the transient absorption as a function of wavelength and time is recorded by spectrally resolving the XUV light after the sample using a spectrometer. A detailed explanation of the experimental apparatus and sample characterization is in the Methods section and 4,5 absorption edge  at different time delays τ are shown in 11 W cm−2, exciting ∼8 × 1020 carriers per cubic centimetre or ∼0.3% of the total number density (see Methods section). This constitutes a high carrier density regime where carrier–carrier scattering effects are expected to play a role. In the time delay span measured from −50 fs to 1.5 ps, where negative delay means the XUV pulse arrives first, transient signals are observed for all time delays. Several features with positive and negative signs in ΔAmeas are observed in the transient absorption signal, with most of the observable dynamics taking place during the first picosecond following excitation. Overlapping spectral features arise from the spin-orbit splitting of the 3d5/2 and 3d3/2 core-level transitions; this splitting, 0.58 eV, is comparable to the bandgap (0.66 eV)254,5 edge   and broadening σ(τ) are estimated. As a convention, here a positive value of ΔEshift(τ) means a redshift of the excited state spectrum. In subsequent iterations, the SB component ΔASB (Eshift(τ) and σ(τ), converging after five iterations. It is found that broadening ΔAbroad plays only a minor role in the experiments presented here and constitutes signals at the noise level     of the sSB  is refinse level . The majift  and the ASB  followind5/2 and 3d3/2 core-level transitions. A Fourier reconstruction method (see Methods section and d5/2). As noted, the SB transient obtained  containsEshift(τ) (In the time-dependent band shift Δshift(τ)  at negatshift(τ) .4,5 edge. Oppositely, the VIS-NIR-excited electrons in the CB block XUV transitions from the core level, leading to a decrease in XUV absorption, and, thus, a negative ΔA. The carrier dynamics features as well as the band shift ΔEshift(τ) rise within the instrumental response time  fs, which is characteristic of the recombination of the carriers, since this is the timescale when the transient carrier dynamics signal vanishes  fs, reversing its sign at the long time limit. This component of the SVD can be interpreted as high-energy electrons (hot carriers) relaxing very rapidly from higher-energy states to fill available states at lower energies within this relaxation time.The carrier dynamics lectrons  are anallectrons  yields glectrons . The comlectrons  can be avanishes . The errvanishes  consistsvanishes  decays w1, Δ1, Γ2′ and weakly L1 stand out with significantly longer lifetimes, because the carriers relax to particular valleys and accumulate there.In addition, single exponential functions were fitted to profiles along the time-delay axis at energies between 29.6 and 30.4 eV , with tiτ increases, representing the hot carrier relaxation and recombination of holes. Decomposing the transient absorption signal by SVD reveals two major components. For the largest component, an assignment is made to a thermalized hole distribution  fs, after the initial excitation and a subsequent τh,recomb= fs decay  fs time constant. This indicates that carrier dynamics, albeit a signal proportional to the population distribution, changes sign at the long time limit. Multiplying the time dynamics and the carrier dynamics component together suggests that the second largest component represents a depletion of hot holes and an increase of low-energy holes as the singular vector in time decays from positive to negative value (The second largest component features a positive distribution at larger (hot) hole energies (∼28 eV) and a negative distribution near the band edge  excitation, which is unprecedented in conventional optical pump-probe studies. The transient absorption of XUV is further decomposed into contributions from electronic SB, band shifts and excited state broadening. To analyse the carrier dynamics in germanium, a Fourier transform-based technique is devised to retrieve the contribution from a single spin-orbit state at the germanium M4,5 edge is generated by high harmonic generation in xenon assisted by polarization-assisted amplitude gating gatingτ between the pulses the transient changes are measured. A shutter periodically blocks the VIS-NIR pump beam to obtain a differential absorption signal. The data set shown in the main text τ= fs. To compensate for time delay drifts in parallel to the germanium sample the differential absorption at the argon 3s3p66p autoionizing state is measuredδE≈0.07 eV from known linewidths in gas absorption spectra. In the postexperimental data analysis, first the measured differential absorption data ΔAmeas data are decomposed into contributions from SB, band shifts and excited state broadening. The high-quality static absorbance used for decomposing these contributions was measured by averaging one hundred transmission spectra alternating between the sample and an empty silicon nitride membrane under otherwise identical conditions to those for the time-resolved measurements. The SB signal is subsequently separated into a single spin-orbit split state by dividing out a constant phase factor in the Fourier domain originating from the spacing  one gets Ne≅8 × 1020 cm−3. Second, the performed TDDFT calculations using k-dependent excitation and the spectrum of the experimental laser pulse an excitation fraction of 0.3% was calculated for a laser intensity of 2 × 1011 W cm−2. Using that a unit cell in germanium has a volume of V0=4.527 × 10−23 cm3 and that there are two atoms or eight valence electrons in this volume, one gets a valence electron density of d0=1.76 × 1023 cm−3. Hence, for the TDDFT-based calculation one gets Ne≅5 × 1020 cm−3.The number of excited electrons has been estimated in two ways. First, from the fluence Eren,therm, a temperature can be assigned to the thin film. For example, for an average pump power of ΔPavg=0.39 mW the measured shift of the edge is ΔEren,therm=72.8 meV, suggesting a temperature of T=475 K, which is confirmed by heat diffusion calculations using a finite element method  sample transmission followed by the excited (hot) sample spectrum to obtain a differential absorption spectrum. A comparison of the absorption change in a heated Ge film to this spectral feature suggests that it is related to an increased temperature of the germanium thin film after optical excitation causing a heat-induced band shiftt method .11 W cm−2 4545cm−2 see .4,5 edge XUV absorption in the presence of excited carriers. The difference between this excited state spectrum and the corresponding ground state spectrum in the absence of electron–hole pairs is compared to the 3d5/2 core-level differential absorption retrieved from experiment at time delays directly after excitation, shown in 4,5 edge absorption to be captured but many-body effects such as excited-state bandgap shifts and carrier lifetime modulation are not described. Therefore, the comparison with experiment is invoked primarily in the context of state blocking where good agreement is observed , with E as photon energy and t the delay time, can be decomposed intoThe carrier dynamics can be decomposed into several carrier distributions with independent relaxation dynamics. Under such formulation, the transient absorption signal Δun is the nth component of transient absorption, which corresponds to a distinct carrier distribution and νn its accompanying relaxation dynamics. sn is the singular value of the nth component, which signifies the importance of the component on the overall dynamics.where ab initio simulation package50The band structure of germanium  is calcuThe experimental data can be obtained on reasonable request from the corresponding authors.How to cite this article: Zürch, M. et al. Direct and simultaneous observation of ultrafast electron and hole dynamics in germanium. Nat. Commun.8, 15734 doi: 10.1038/ncomms15734 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Notes and Supplementary References


Natalizumab (NTZ) is a drug that has been widely used in the treatment of multiple sclerosis (MS). NTZ is very effective in suppressing inflammation, but if treatment is suspended many patients will experience relapses.+ NK cells could predict early disease reactivation after NTZ suspension.To investigate if quantification of γδ T cells and HLA-DR+ NK cells in whole blood were determined with flow cytometry in fifteen patients treated with NTZ. NTZ treatment was then withdrawn and patients were followed with clinical visits and MR investigations.Absolute counts of γδ T cells and HLA-DR+ NK cells.Patients with recurrent disease had higher absolute counts of γδ T cells 129 (±156) cells/μl in comparison to patients with stable disease 50.0 (±51.0) cells/μl but the difference was not statistically significant and largely driven by outliers. Patients with recurrent and stable disease had similar absolute counts of HLA-DR+ NK cells could not predict active disease after NTZ suspension.Quantification of γδ T cells and HLA-DR Multiple sclerosis (MS) is a severe neurological disorder leading to functional impairment and disability. At onset, most patients have a relapsing-remitting disease with periods of loss of function (relapses) followed by recovery. Such relapses are caused by localized inflammation in the brain and spinal cord, leading to disruption of neuronal signaling and tissue damage.In the last two decades, several drugs have been developed to suppress and modulate inflammation in MS. Considerable effort has been put into proving the efficacy of these drugs, less so whether and when it is time to stop treatment. From natural history studies it is known that inflammation in MS tends to diminish with age . ConsequNTZ is a monoclonal antibody that binds to α4β1 integrin on leukocyte plasma membranes, which prevents the interaction between α4β1 integrin and its cognate ligand vascular cell adhesion molecule-1 (VCAM-1). This blocking prevents extravasation of leukocytes to the effect that the leukocytes are trapped in the peripheral circulation and thus prevented from entering and damaging the central nervous system . A serio+ NK cells, which can be readily quantified with standardized assays. We hypothesized that quantification of these in patients treated with NTZ would be able to predict subsequent relapses after discontinuation of natalizumab. To test this hypothesis we quantified γδ T cells and HLA-DR+ NK cells in blood samples from patients who were participating in a clinical trial investigating whether NTZ could be safely withdrawn after long-term treatment [+ NK cells from a group of patients commencing NTZ therapy.Since pathogenic lymphocytes are trapped within the peripheral circulation with NTZ treatment, it would be enticing to see whether it is possible to detect and quantify these in order to predict subsequent relapses after NTZ withdrawal. In a study made some years ago, a large number of immune subsets were investigated and related to disease activity . The invreatment . In ordeThe study was approved by the Ethics Committee of the Medical Faculty of Uppsala University (DNr 2013/293). All subjects provided written informed consent.+ NK cells. Therefore it was decided to include 20 patients in the study.A power calculation was made based on based on data from Rinaldi et al . We assu+ NK cell subsets after initiation of NTZ therapy seven patients commencing NTZ treatment were included. Their characteristics are summarized in To investigate possible changes in the γδ T cell and HLA-DRth 2014 and the last patient was included October 20th 2014. Further recruitment to the study was halted for ethical reasons when a significant portion of the first 15 patients experienced severe relapses.Patients who were participating in a clinical trial investigating whether NTZ could be safely withdrawn after long-term treatment  were recPatients commencing NTZ treatment were evaluated for disease activity at a follow-up visit at the end of the study (after six months).Prior to entering the study, patients suspending NTZ were followed with scheduled clinical visits with EDSS scoring twice a year. Brain MRI was performed yearly. At baseline all patients had an MRI scan of the brain and spinal cord and MRI was repeated at three and six months. Patients were instructed to contact the study nurse if symptoms consistent with a relapse occurred. If so, an extra clinical visit was scheduled and an extra MRI was made.Patients who had at least one gadolinium-enhancing lesion on any MRI scan performed at any time point after NTZ suspension were considered to have active disease.Ten ccs of venous blood were drawn from patients starting NTZ at baseline and then again three and six months after treatment commenced.Ten ccs of venous blood were drawn bi-monthly starting at six months before suspension of NTZ treatment . After NTZ suspension further blood samples were taken at three and six months on the same day as the MRI scans. Some patients experienced severe relapses before follow-up at 6 months and started alternative treatment. Those patients did not leave blood samples at 6 months.+ NK cells was quantitated with flow cytometry in fresh whole blood samples. Measurements of absolute counts of T cells and NK cells were made with BD Truecount Tubes . The antibodies used are detailed in + CD4- CD8- γ/δ TCR+ lymphocytes. HLA-DR+ NK cells were defined as CD3- CD16+ CD56+ HLA-DR+ lymphocytes. The cells were acquired on a BD FACS Canto II. Flow cytometry data were analyzed with FlowJo v 10.1 .The number and proportions of γδ T cells and HLA-DRThe statistical analysis was carried out with GraphPad Prism 6 . The Mann-Whitney test was used to establish statistical significance between two groups and the Friedman test was used to establish statistical significance between different time points within the same group. A p-value of 0.05 was considered to be statistically significant. Aggregated data is described as mean ± standard deviation (SD) or as median with range.After the initiation of NTZ treatment, all patients were stable and did not experience clinical relapses or increase in disability.Six out of fifteen patients had active disease with gadolinium-enhancing lesions on MRI within the first six months after suspending NTZ treatment and all of them also had a clinical relapse .+ NK cell subsets change with NTZ treatment. After the introduction of NTZ there was a statistically significant increase in γδ T cell count (p = 0.0036) after 6 months of the treatment (μl at baseline to 41.6 (±33.4) cells/μl 6 months after NTZ initiation. The proportion of γδ T cells in the total T cell population remained stable over the period of observation for most patients  (+ NK cell counts increased from 25.3 (±13.3) cells/μl at the baseline to 55.3 (±21.2) cells/μl 6 months after NTZ initiation. The proportion of HLA-DR+ NK cells in the total NK cell population was fairly stable  . The aveable see .+ NK cells populations were stable in patients who had been treated with NTZ for a long time. The absolute counts of γδ T cells remained fairly constant over a period of six months  cells/μl after the cessation of NTZ, but the difference was not statistically significant (p = 0.70). The proportion of γδ T cells in the total T cell population remained unchanged after the suspension of NTZ (The absolute counts of γδ T cells decreased in a majority of patients after the cessation of NTZ  and the n of NTZ .+ NK cells decreased after the termination of NTZ (+ NK cells decreased from 46.1 (±37.5) cells/μl at baseline to 23.8 (±15.3) cells/μl after the suspension of NTZ, which was borderline statistically significant (p = 0.08). The proportion of HLA-DR+ NK cells in the total NK cell population remained unchanged  cells/μl and stable disease 50.0 (±51.0) cells/μl which was not statistically significant and largely driven by outliers . The sam+ NK cells were fairly similar in all patients  % for active patients and 18.7 (±14.7) % for stable patients , suggesting that there may be a biologically relevant difference between active and stable patients in this immune subset. However, the interindividual variation of this metric was too high to be useful as a clinical test.In the second part of the study, we tried to determine if it is possible to predict who will get active disease after withdrawal of NTZ treatment. This part of the study was largely based on the results of Rinaldi et al. . The val+ NK cells . This discrepancy may be due to differences in methodology; γδ T cells constitute an easily identified subpopulation of T cells and their quantification is very straightforward. In contrast, the expression of HLA-DR on NK cells is gradual and the classification of NK cells as HLA-DR+ or HLA-DR- is therefore more difficult and somewhat subjective. Another possible explanation for the disparate findings is that NTZ affects NK cells in a hitherto unknown manner, which is different from the effect on T cells. Underlying differences in the studied cohorts, such as age or previous exposure to immunotherapy, may also have influenced the results.We could not confirm the findings of Rinaldi et al. in the frequencies of HLA-DROther strategies to measure immunological disease activity may be more successful. In a recent study it was suggested that a low frequency of Th17 cells in blood could act as a marker for MS relapse after the discontinuation of NTZ . Patient+ NK cells increase with NTZ treatment, that the levels are fairly stable during treatment and that they decrease when NTZ treatment is withdrawn. Furthermore, we could demonstrate that the proportion of γδ T cells and HLA-DR+ NK cells are essentially unchanged with NTZ treatment. However, the main objective was negative and we conclude that the quantification of γδ T cells and HLA-DR+ NK cells does not predict active disease after NTZ suspension.In summary, we could confirm the expected findings that the absolute counts of γδ T cells and HLA-DRS1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.


The SC protocol using larvae generates animals with sustained production of modified sperm , but is a time-consuming protocol . GT is a faster technique, but the modified gamete production is temporary . ET is an easier way to obtain mosaic transgenic animals compared to microinjection of eggs, but non-site-directed integration in the fish genome can be a problem. In this study, PI3Kc2α gene disruption impaired development during the embryo stage and caused premature death. The manipulator should choose a technique based on the time available for transgenic obtainment and if this generation is required to be continuous or not.Microinjection is commonly performed to achieve fish transgenesis; however, due to difficulties associated with this technique, new strategies are being developed. Here we evaluate the potential of lentiviral particles to genetically modify Nile tilapia cells to achieve transgenesis using three different approaches: spermatogonial stem cell (SSC) genetic modification and transplantation (SC),  Oncorhynchus mykiss) by microinjecting newly fertilised eggs34in vivo transduction of spermatozoa in adult fish testis, and in vitro genetic modification of spermatogonial stem cells (SSCs) followed by their transplantation into Nile tilapia larvae. Researchers have used these techniques to generate transgenic mice and rats678et al., the authors used a retrovirus and did not show the integration site or mention any types of damages owing to the genomic integration. Lentiviruses can integrate in regions that encode gene products. This integration can cause problems by disrupting some important genes, but it can also be used as an important tool for studying gene function by observing the effects of insertional mutagenesisFish transgenesis has received attention since the early 1980s, when Mclean and Talwar genetically modified rainbow trout , eye region (d), connective tissue (e), and skeletal muscle , which is in accordance with the fact that the promoter used in DNA construction was the CMV promoter, a strong and non-tissue-specific promoter.Through immunohistochemical assay, it was observed that the larvae developed from eggs exposed to the maximum lentiviral concentration presented a disseminated DsRed2 expression, as illustrated in the blood cells c, eye ree region d, connece tissue e, and skPI3Kc2α)  . In fishPI3Kc2α) .The fish that suffered premature death (~67%) died from bleeding . ImmunohThe second genetic modification strategy was performed in SSCs exposed to lentiviral particles. Isolated Nile tilapia SSCs can be phenotypically identified by the expression of some early germ cell markers such as Vasa and Nanos2et al. 2006, Farlora et al. 2014, Morita et al. 2015). Seven days after intracoelomic injection of transduced SSCs, it was already possible to observe fluorescence related to the transduced and transplanted cells near the recipient genital ridge area  gene. This helps explaining why fish that suffered premature death (~67%) died from bleeding . The enzin vivoImpairment in the growth of DsRed2 fish could also be caused by the interruption of the PI3Kc2α gene. In mouse embryos, lack of this enzyme causes defects and eventually lethality as a result of defective vasculogenesisThese findings pave the way for future studies of this enzyme’s overexpression or co-expression with growth hormone (GH) to generate a transgenic fish that grows faster than expected. Some transgenic animals overexpressing GH present impairment of cardiac functionThus, the GH-PI3Kc2α DNA construct may offer better transgenic fish with sustained angiogenesis to achieve growth in a more stable way. Overexpression of PI3Kc2α alone may be able to create transgenic fish with faster growth and without unfavourable phenotypes commonly observed under hypoxia.in vivo transduction strategy, 70% of the animals presented genetic modification of the sperm, and the maximum fluorescence achieved was approximately 52%; values lower than those obtained through SSC transplantation .Using the in vitro fertilization), also results in animals with sustained production of modified gametes. Both methodologies mentioned work well in producing transgenic gametes, but in vivo transduction results in fish that cannot maintain a high level of fluorescence in the sperm, like the strategy involving SSC in vitro manipulation  transgenic sperm in comparison to the SSC strategy; and also with fluorescent sperm whose production is only temporary in fish, as one week following the first analysis the sperm fluorescence was drastically reduced (from ~52% to ~9%).In this study, we evaluated three different methodologies for genetically modifying Nile tilapia . AlthougPI3Kc2α gene disruption in fish: impaired development during the embryo stage, premature death, and blood vessel compromise were present in Nile tilapia, as previously reported in the literature for mice, when the insertion of lentiviral particle genetic material occurred inside the kinase encoding sequence. This paves the way for the study of the effects of PI3Kc2α overexpression or co-expression with the GH encoding sequence to generate transgenic fish, supporting increased growth and fast development in a more favourable and stable way and avoiding impairment of cardiovascular function of animals under low oxygen tension.The results also made it possible to observe the effects of Escherichia coli (INVITROGEN). The transformants were selected using ampicillin (100 μg/mL), and 19 colonies were submitted to colony PCR in order to verify their insert orientation (the primers used were V5 and FWD R1). The ones containing the DsRed2 encoding sequence in correct orientation were expanded and their plasmid DNA extracted using the PureYield Plasmid Maxiprep System (PROMEGA). The presence of the desired encoding sequence was confirmed by DNA sequencing using the primers CMV and V5, BigDye Terminator v3.1 Cycle Sequencing (INVITROGEN), and Hi-Dye formamide (INVITROGEN) in a DNA ABI 3130 automatic sequencer (APPLIED BIOSYSTEMS). The lentiviral particles were generated by cotransfection into 293FT producer cells (INVITROGEN) with components of the ViraPower Lentiviral Expression System (INVITROGEN) according to the manufacturer’s protocol. The titre of lentiviral particles was determined through transduction of HeLa cells in the presence of Polybrene (SIGMA).The lentiviral particles were obtained using the pLenti6.3/V5-TOPO TA Cloning Kit (INVITROGEN). According to the manufacturer’s protocol, the encoding sequence for DsRed2 fluorescent protein was inserted into pLenti6.3/V5-TOPO. This sequence was amplified by PCR from pDsRed2 (CLONTECH) using the primers CCGCGGATGGCCTCTTTGCTGAAG-FWD R1 and GGGCCCTCAGTTGTGGCCCAGCTT-REV R1 (56 °C) and DreamTaq DNA Polymerase (THERMO). The construct was verified with primers V5 (ACCGAGGAGAGGGTTAGGGAT) and CMV (CGCAAATGGGCGGTAGGCGTG) from the commercial kit and used to transform the quimiocompetent One Shot Stbl3 In order to verify that lentiviral particles were able to induce the generation of animals with genetic modification of the gonad after being in contact with newly fertilised eggs, we also developed a DNA construct containing the sequences to generate by the same protocol lentiviral particles like those previously presented but containing the codifying sequence of eGFP fluorescent protein under the control of a germline specific promoter: the Vasa (pLenti-VseGFP).5 TU–1000 μL, 3.0 × 105 TU–500 μL, 1.8 × 105 TU–300 μL, 1.5 × 105 TU–250 μL, 6.0 × 104 TU–100 μL), for 24 h under agitation at 28 °C . After this time, the eggs were washed with PBS and transferred to nylon sieves under constant oxygenation in tank water at the same temperature. The expression of the red fluorescent protein was assessed by fluorescence microscopy in fish from 7 to 20 d of age. Five 20-day-old fish that developed from eggs at each of the tested concentrations had their RNA extracted using Trizol (THERMO). The presence of the DsRed2 encoding sequence was analysed by RT-PCR (first strand synthesised using the oligo(dT) primer, and the second reaction performed with primers to the DsRed2 sequence . Immunohistochemistry was performed in Nile tilapia larvae using Living Colors Anti-RCFP Polyclonal Pan Antibody  and Goat anti-Rabbit IgG HRP conjugate  according to protocol previously establishedThirty eggs of Nile tilapia were exposed to DsRed2 lentiviral particles in Petri dishes with tank water at five different concentrations . The cells were incubated at 33 °C and 5% CO2 for 24 h, and the aliquot was submitted to flow cytometry in order to assess the percentage of fluorescent cells before selection. Blasticidin (6 μg/mL) was added to the supplemented DMEM/F12 culture medium (High Glucose DMEM/F12 (THERMO), 10% foetal bovine serum, 0.1 mM non-essential amino acids, 6 mM L-glutamine, 1 mM sodium pyruvate, and 1% penicillin-streptomycin) and the cells were maintained for one month4 SSCs/μL, using a micromanipulator (NARISHIGE) and glass needles prepared with GD-1 capillaries (NARISHIGE) as previously describedNile tilapia SSCs were obtained through primary culture from adult fish testis1222235 TU) through the common spermatic duct using a glass micropipette (outside diameter 70 μm) under a stereomicroscope (OLYMPUS SZX-ILLB2-100)In adult Nile tilapia, the only non-surgical access to the testis is via the common spermatic duct that opens in the urogenital papilla through the urogenital pore. Ten 6-month-old male fish were anesthetised and received 300 μL of DsRed2 lentiviral particle solution .How to cite this article: Tonelli, F. M. P. et al. Gene delivery to nile tilapia cells for transgenesis and the role of PI3K-c2α in angiogenesis. Sci. Rep.7, 44317; doi: 10.1038/srep44317 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Therefore, we expect this framework to have numerous applications in drug and protein design.TEM β-lactamase confers bacteria with resistance to many antibiotics and rapidly evolves activity against new drugs. However, functional changes are not easily explained by differences in crystal structures. We employ Markov state models to identify hidden conformations and explore their role in determining TEM’s specificity. We integrate these models with existing drug-design tools to create a new technique, called Boltzmann docking, which better predicts TEM specificity by accounting for conformational heterogeneity. Using our MSMs, we identify hidden states whose populations correlate with activity against cefotaxime. To experimentally detect our predicted hidden states, we use rapid mass spectrometric footprinting and confirm our models’ prediction that increased cefotaxime activity correlates with reduced Ω-loop flexibility. Finally, we design novel variants to stabilize the hidden cefotaximase states, and find their populations predict activity against cefotaxime  Expression of TEM β-lactamase is a predominant mechanism underlying antibiotic resistance in pathogenic Gram-negative bacteria. Here, the authors use Markov state models to reveal and experimentally confirm hidden conformations that determine TEM substrate specificity. Antibiotic resistance is a global health threat that results in millions of deaths and billions of dollars in health-care costs every yearUnlike enzymes where conformational changes are known to be important for function677We hypothesize that hidden conformations not yet captured by traditional structural techniques are the missing ingredients required to connect TEM’s structure with function and to predict the effects of mutations. This hypothesis is supported by computational models and room temperature crystals that have revealed TEM adopts diverse structures111213151618192021423in vitro and in vivo.Here, we employ Markov state models (MSMs)2628292324If the ability of ground-state structures of TEM variants to bind different substrates specifies their activities, then it should be possible to make predictions by docking substrates against crystal structures, when available, or homology models when structures have not yet been solved. To test this hypothesis, we chose to study a series of variants with differing activities against cefotaxime, a substrate for which new activity evolved in the clinic. First, we built homology models for the variants. Although there are crystal structures for many different variants of TEM, this is not true for other enzymes we might wish to study. To preserve the generalizability of this method, we chose to build all the homology models using TEM-1 ref.  to mimickcat/Km values. In fact, we observe that the docking scores and activities are anticorrelated . These failed predictions could be due to shortcomings in the force field, which describes the atomic interactions used to produce a docking score. However, they could also be interpreted as evidence for the inadequacy of focusing on single, rigid structures when we know that proteins actually adopt a distribution of different structures at thermal equilibrium. Indeed, we have previously demonstrated that TEM β-lactamase adopts a range of different conformations12Docking against single structures of each variant fails to predict their abilities to degrade cefotaxime, as measured by their To test whether considering proteins’ hidden states is crucial for predicting their functions, we developed a technique called Boltzmann docking that approximates compounds’ relative binding affinities by calculating the ensemble-average score across a set of structural states, weighting each state by its equilibrium probability. Our approach differs from existing ensemble docking methods, which dock compounds against a set of structures sampled via molecular dynamics simulations and then rank the compounds based on the highest score against any of the target structures3334To perform Boltzmann docking, we first built an MSM for each variant based on 2.5 μs of atomically detailed molecular dynamics simulations. Then we docked cefotaxime against a representative structure from each state and calculated an average score, weighting the contribution of each state by its equilibrium probability according to the MSM. More details are given in Methods section.R=0.30±0.10 versus –0.37±0.07). For comparison, we also performed classical ensemble docking, which ranks compounds based on their highest score against any single conformation from a set of structures. We find a correlation coefficient between ensemble docking and experiments of R=−0.02±0.07, indicating no correlation. Thus, while ensemble docking is superior to docking against single structures, which shows an anticorrelation, population-based averaging with Boltzmann docking is the only method that results in a physically meaningful correlation with experiments. Interestingly, the highest scoring docking poses for both wild type and E104K/G238S bind to structures that closely resemble the ligand-free crystal structures   . It was To determine how the E104K/G238S substitutions alter the specificity of TEM, we constructed MSMs for both wild type and E104K/G238S. We used the crystal structure of wild typeTo identify states that may be responsible for degrading cefotaxime, which we call cefotaximase states, we queried the MSMs for all states that are significantly more populated by the E104K/G238S variant, which can degrade both cefotaxime and benzylpenicillin, over wild type, which can only degrade benzylpenicillin effectively. We also identified structures that are more populated by wild type than E104K/G238S, which we refer to as non-cefotaximase states. This analysis reveals that the Ω-loop of wild type undergoes substantial rearrangements that are absent in E104K/G238S . The Ω-l7If the conformational preferences and amplitudes of the Ω-loop’s fluctuations are key determinants of the different specificities, then we should be able to predict the activities of other variants based on the properties of their Ω-loops. Variants that populate the cefotaximase states should have higher activities against this substrate than variants that preferentially populate the non-cefotaximase states. Because the single G238S substitution confers substantial resistance to cefotaxime but the E104K substitution does not, we expect G238S to resemble E104K/G238S, whereas E104K should more closely resemble wild type. Consistent with our hypothesis, the G238S substitution populates the cefotaximase states more than wild type and E104K but less than E104K/G238S . E104K pExamining the distribution of structures reveals that each substitution pins down the side of the Ω-loop to which it is adjacent. G238S appears to do this by hydrogen bonding with residues in the Ω-loop. For example, it forms a hydrogen bond with the carbonyl of Asn170 in 70% of the population . E104K aα positions of Glu240 in G238S-containing structures versus TEM-1), which results in a small widening of the active site. In contrast, we observe that both loops assume conformations more similar to the wild-type crystal structure in our most populated cefotaximase states  has been observed in directed evolution studiesin vitro activities against cefotaxime and the extent to which they confer cefotaxime resistance to E. coli . Furthermore, the correlation is robust to exclusion of the variant used to define cefotaximase states, E104K/G238S (R=0.74±0.04). This result supports our conclusion that MSMs provide a reasonably accurate depiction of TEM β-lactamases’ structural ensembles. Boltzmann docking performed better than docking against single structures by accounting for conformational heterogeneity, but any docking approach is limited by using substrate binding as a proxy for activity. Enzymes undergo conformational changes throughout a catalytic cycle in concert with chemical transformations to the substrate, so these states would not necessarily score well. MSMs, on the other hand, may capture these catalytically-relevant states. Therefore, a powerful approach to classify the activities of new variants is to compare MSMs of their structural ensembles to MSMs for variants with known functions.Impressively, the populations of our hidden cefotaximase states provide the most accurate predictions of cefotaxime activity . A long-range dispersion correction was employed for both energy and pressure. All bonds were constrained with the LINCS algorithmFive 500 ns simulations were run for each variant with Gromacs 4.6.5 ref.  and the 12MSMs were constructed with MSMBuilder (v2.8)28MSMs for comparing the structural preferences of different variants were constructed based on the same set of active-site residues. First, every 100th data point from simulations of each variant were pooled together and clustered into 1,000 states with a k-medoids algorithm. Then the equilibrium probability of each state for a given variant was calculated using the same approach described before using just the data for that variant. Using a common set of states to describe the thermodynamics and kinetics of each variant provides a basis for directly comparing the probabilities that different variants will adopt a given conformation. Of the 1,000 states in this combined state space, we found that 350 of these states have higher populations in E104K/G238S than wild type. As explained in the text, we designate these states as cefotaximase states and employ the sum of the equilibrium populations of these states  as a surrogate for cefotaxime activity. Another 399 states are more populated by wild type than E104K/G238S, which we designate as non-cefotaximase states.n−1 data points (where n is the number of TEM variants) and reporting the mean and standard deviation of these correlation coefficients. Implied timescales were also obtained by calculating the implied timescales of all n possible subsets of n−1 trajectories (where n is the number of independent simulations) and reporting the mean and standard deviation of these timescales.Error bars on the population of a subset of MSM states were obtained via bootstrapping. That is, we drew 100 independent subsamples of the data (with replacement) and reported the mean and standard deviation of the sum of the equilibrium probabilities of the subset of states of interest. Correlation coefficients between the populations of subsets of MSM states and experiments were obtained by calculating the Pearson correlation coefficient between all n possible subsets of Inter-atomic distances were calculated with MDTrajDocking against individual structures was performed with Surflex-dockBoltzmann docking was performed using the same settings to dock the substrates against the cluster centres from each state of the MSMs built for an individual variant. The final score was then calculated as the weighted-average of the scores for each state, using the equilibrium probabilities of each state as their weights. Ensemble docking was conducted by taking the same set of scores against cluster centres and ranking compounds based on their highest score against any of the protein structures.Correlation coefficients between different docking protocols and experiments were calculated in the same manner as the correlations between the populations of subsets of MSM states and experiments, except that we compare to the log of the experimental enzyme efficiencies to put these measurements on an energy scale, like the docking score.TEM-1 was subcloned using NdeI and XhoI restriction sites into the multiple cloning site of a pET24 vector (Life Technologies), and its native export signal sequence was replaced by the OmpA signal sequence to maximize export efficiency4 and stirred for 10 min at 4 °C. After centrifugation, the supernatant was dialyzed against 20 mM sodium acetate, pH 5.5 and purified using cation exchange chromatography (BioRad UNOsphere Rapid S column) followed size exclusion chromatography (BioRad ENrich SEC 70 column) into storage buffer .Cells were induced with 1 mM IPTG at OD=0.6 and grown at 18 °C for 15 h before harvesting. TEM β-lactamases were isolated from the periplasmic fraction using osmotic shock lysis: Cells were resuspended in 30 mM Tris pH 8, 20% sucrose and stirred for 10 min at room temperature. After centrifugation, the pellet was resuspended in ice-cold 5 mM MgSO2O2, as described originallyL-glutamine and 15 mM H2O2 at room temperature and flowed in a 150 μm i.d. capillary by a syringe pump  at a rate of 24 μl min−1 past a transparent laser window where the flowing solution was irradiated by 248 nm KrF excimer laser . The laser power was adjusted to ∼35 mJ pulse−1 at a frequency of 7 Hz, ensuring an exclusion volume fraction of 20% having no laser irradiation. The laser-irradiated sample was collected in a vial containing 500 nM catalase and 70 mM L-methionine to decompose excess H2O2. The sample was frozen in liquid nitrogen and then stored in −80 °C until further analysis.TEM variants were submitted to FPOP by irradiating with an excimer laser pulse on the flowing mixture of protein and HFor each mutant the labelling reaction was done in triplicate. The sample was divided into two parts. One part was analysed using a Bruker Maxis Q-TOF mass spectrometer  under denaturing conditions to check for hydroxyl radical labelling at the protein level. The other was treated with 20 mM TCEP, PBS, pH 7.4 at 45 °C for 1 h to reduce the disulfide bond in PBS pH 8.0. The alkylated protein sample was immediately purified using the 2-D clean-up kit , denatured in presence of 8 M urea and digested overnight with trypsin in 100 mM TEAB, pH 7.8 at 37 °C. The resulting peptides were separated by reversed-phase HPLC using a Dionex Ultimate 3,000 RSLCnano system  and introduced to a Thermo QExactive Plus mass spectrometer  via nano-ESI. Peptide ions were fragmented in a data-dependent mode in which the 10 most abundant components were selected by the spectrometer for fragmentation. Unmodified and modified peptides were identified by Mascot  and Byonic  and confirmed by manual inspection.εBP=−1,096 M−1 cm−1) or 265  using a Cary 100 UV–vis spectrophotometer (Agilent Technologies). Velocities were plotted as a function of substrate concentration and fit by the Michaelis–Menten equation to extract kcat and Km values. Enzymes that did not exhibit saturation behaviour under the tested conditions were fit by a line, and the slopes are reported as kcat/Km.Enzyme activities against benzylpenicillin (BP) and cefotaxime (CFX) substrates were measured in 50 mM potassium phosphate, 10% glycerol (v:v) pH 7.0 at 25 °C using 2, 10 or 200nM enzyme. The reaction was monitored at 232 nm (Site-specific variants of TEM-1 were constructed via site-directed mutagenesis of the pBR322 plasmid and verified by DNA sequencing. Plasmids were then transformed into BL21(DE3) cells (Intact Genomics) and DH5α cells (Life Technologies) to create strains in which β-lactamases are expressed using a native promoter.5 c.f.u. ml−1) was then added to each well giving a total volume of 150 μl per well and compound concentration gradients of 48 mM–23 μM (BP) and 72–0.04 μM (CFX). The plate was incubated at 37 °C for 17 h, and then each well was examined for bacterial growth. The MIC90 was recorded as the lowest compound concentration required to inhibit 90% of bacterial growth as judged by turbidity of the culture media relative to a row of wells filled with a water standard. Gentamicin was included in a control row at a concentration gradient of 174–0.09 μM.Antibiotic resistance of the strains was determined by measuring their minimum inhibitory concentrations (MIC90’s) using the broth microdilution method according to the Clinical and Laboratory Standards Institute  guidelinesThe experimental data supporting the findings of this study as well as the computer code and simulations are available from the corresponding author upon request.How to cite this article: Hart, K. M. et al. Modelling proteins’ hidden conformations to predict antibiotic resistance. Nat. Commun.7, 12965 doi: 10.1038/ncomms12965 (2016).Supplementary Figures 1 - 4


Li+ (turquoise), the better charge acceptor, can displace Na+ (purple) bound by only one or two aa residues in buried sites. Thus, Li+ can displace Na+ bound by Asp– and Ser in the A2AAR/β1AR receptor and enhance the metal site's stability, thus prohibiting structural distortions induced by agonist binding, leading to lower cytosolic levels of activated G-proteins, which are hyperactive in bipolar disorder patients. +) acts as an indispensable allosteric regulator of the activities of biologically important neurotransmitter transporters and G-protein coupled receptors (GPCRs), which comprise well-known drug targets for psychiatric disorders and addictive behavior. How selective these allosteric Na+-binding sites are for the cognate cation over abiogenic Li+, a first-line drug to treat bipolar disorder, is unclear. Here, we reveal how properties of the host protein and its binding cavity affect the outcome of the competition between Li+ and Na+ for allosteric binding sites in sodium transporters and receptors. We show that rigid Na+-sites that are crowded with multiple protein ligands are well-protected against Li+ attack, but their flexible counterparts or buried Na+-sites containing only one or two protein ligands are vulnerable to Li+ substitution. These findings suggest a novel possible mode of Li+ therapeutic action: By displacing Na+ bound by ≤2 protein ligands in buried GPCR sites and stabilizing the receptor's inactive state, Li+ could prohibit conformational changes to an active state, leading to lower cytosolic levels of activated guanine nucleotide-binding proteins, which are hyperactive/overexpressed in bipolar disorder patients.Sodium (Na Several hypotheses have been put forth, most of which focus on the competition or cooperation between Li+ and native ions such as Mg2+ and Na+ in biological compartments.Lithium  and guanosine triphosphate (GTP), which act as activators of signal-transducing proteins.2+ channel), has been shown to elicit longer P2X activation than the native ATP–Mg cofactor.+ binding to [ATP–Mg]2– does not alter the native cofactor's overall charge, conformation, and interactions with protein ligands lining the P2X binding site.+ in its preferred solution mode, then Li+ binding to ATP–Mg could reduce the native cofactor's susceptibility to hydrolysis, resulting in prolonged residence of the bimetallic ATP–Mg–Li complex in P2X and thus, prolonged receptor response.+ binding to [GTP–Mg]2– results in lower cytosolic levels of activated G-proteins and eventually normalizes cell signaling.14Instead of competing with Mg+, in its free hydrated state, can compete with Na+. Bipolar disorder patients have abnormally high Na+ cytosolic concentrations.+, by entering the cytosol via sodium channels, accumulates inside the cell lowering the intracellular concentration of Na+, which in turn reduces the Ca2+ cytosolic level. Decreasing the intracellular concentrations of both Na+ and Ca2+ attenuates the cell excitability, eventually normalizing the neuronal activity in bipolar disorder patients.15Another hypothesis posits that Li+ therapeutic action has not been considered (to our knowledge); viz., the competition between Li+ and Na+ for specific Na+-binding sites in signal-transducing proteins, notably neurotransmitter transporters and G-protein coupled receptors (GPCRs), which comprise well-known drug targets for psychiatric disorders+, which acts as an indispensable allosteric regulator of their activities.+ allosteric control.+ and Na+ in these systems have not been addressed:However, another possible mode of Li+ over Li+ in Na+-proteins?(1) What are the key determinants controlling the selectivity for Na+-binding sites are more vulnerable to Li+ substitution? If Li+ can displace Na+ from its binding site, how would this affect the host protein function?(2) Which Na+-binding sites to determine their characteristic features. Then, we evaluated the thermodynamic outcome of the competition between Na+ and Li+ in various model transporter/receptor Na+-binding sites, as described in Methods. We reveal how this outcome depends on properties of the host protein and its binding cavity and which Na+-sites are prone to Li+ substitution. Our findings suggest a novel possible mode of Li+ therapeutic action.Here, we aim to answer these questions. First, we surveyed the Protein Data Bank (PDB)+; the resulting PDB entries are listed in ESI Table S1.+-bound ligands were identified by a cutoff of 3 Å. If the same protein has more than one structure, the highest-resolution one with the most number of Na+-bound ligands was chosen. This yielded 16 representative proteins  of the residue X in the protein to the accessible surface area of X in the tripeptide –Gly–X–Gly– using the MOLMOL+-ligating residues using the HBPLUS Hydrogen Bond Calculator version 3.15.+-ligating residues in the representative PDB structures are listed in ESI Table S2.The PDBproteins  with dis+-sites from the protein crystal structures, the Na+-binding sites (without the substrate) were modeled as mononuclear penta/hexacoordinated complexes, comprising mostly neutral O-containing protein ligands (see N-Methylacetamide (CH3CONHCH3) was used to model the backbone peptide group as well as the Asn/Gln side chain rather than acetamide (CH3CONH2), as replacing a H from the acetamide NH2 with a methyl group does not alter metal binding and N-methylacetamide and acetamide have similar solvation free energies.–/Glu– side chains were modeled as ethanol (CH3CH2OH) and acetate (CH3COO–), respectively.On the basis of the structure/composition of Naab initio methods/density functionals  and basis sets , 6-31+G, 6-31+G, 6-31+G, 6-311++G and 6-311++G), the B3-LYP/6-31+G method has been found to be the most efficient in reproducing experimental observables: it could reproduce  the dipole moments of water and protein ligands,+, K+, Li+, Mg2+, Ca2+, Fe3+, and Ga3+) in aqua and crown ether complexes+, Na+, K+, Mg2+ and Ca2+) exchange in crown ether, acetate, oxalate and nitrilotriacetic acid complexes.Eel, using the Gaussian 09 program.51Among various combinations of +. During geometry optimization, some hexacoordinated Na+ complexes isomerized to more stable pentacoordinated structures with a ligand relegated to the second shell. These were retained for further evaluation. The fully optimized Na+ complexes were used as starting structures for optimizing the respective Li+ counterparts. In the fully optimized Li+ complexes, two ligands spontaneously moved to the metal's second shell, leaving four ligands coordinated to Li+, consistent with lithium's known preference for tetrahedral complexes.Initially, we built hexacoordinated complexes with all six protein and/or water ligands bound to NaGε for eqn (1)+-protein] and [M+-aq] (M+ = Li+ or Na+) represent the cation bound to protein ligands in the binding cavity and unbound in its vicinity, respectively. The cation exchange ΔGε in a binding cavity characterized by an effective dielectric constant ε can be evaluated asG1 is the gas-phase free energy for eqn (1) and ΔGsolvε is the solvation free energy ΔGsolvε. To mimic varying solvent exposure of the metal-binding cavity, ε was varied from 4 to 30.Based on the fully optimized structures, we computed the free energy ΔEel), thermal energies (ΔEth) and entropies (ΔS) between the products and reactants in eqn (1) at a temperature T of 298.15 K according to:This was computed from the differences in the electronic energies (Δ–1),In evaluating the thermal energies and entropies, the vibrational frequencies were scaled by an empirical factor of 0.9613.+, Na+, and model ligands to within 1.1 kcal mol–1.RLi = 1.38, RNa = 1.70, RC = 1.95, RN = 1.75, RO(–CONH–) = 1.72, RO(–CH2OH) = 1.90, RO(H2O/Na–H2O) = 1.85, RO(Li–H2O) = 1.84, RO(COO) = 1.40, RH = 1.50, RH(Na–H2O) = 1.26, and RH(Li–H2O) = 1.44.This was computed by solving Poisson's equation with natural bond orbital atomic charges+-binding sites: sodium transporters generally possess two or more relatively “dry” juxtaposed Na+ sites where each Na+ is bound by mostly protein ligands rather than water molecules. In contrast, sodium receptors exhibit only one Na+-binding site, where the Na+ is generally bound by fewer (≤3) protein ligands and up to four water molecules.The results in + as well as the relative burial and flexibility of allosteric Na+-binding sites. A Na+-binding site is deemed to be buried if the average relative SASA of the Na+-ligating amino acid (aa) residues is <20%, partially buried if it is between 20 and 50%, and solvent accessible if it is >50%  Na+ is generally penta/hexacoordinated in receptors and transporters, albeit for structures with resolution >2.5 Å, the Na+ coordination number is uncertain since bound water molecule(s) may not be seen in the structure.(b) Na+, being a “hard” cation, binds to “hard” oxygen atoms from the protein ligands.(c) Na+ is bound by at least two aa ligands and no more than two negatively charged ones.(d) Na+ is preferentially bound by neutral protein ligands  rather than negatively charged ones (Asp–/Glu–), which generally bind Na+via one rather than both carboxylate oxygen atoms.(e) Na+-binding sites in both transporter and receptor proteins are generally quite buried with multiple (≥3) hydrogen bonds  Na+ with Li+ in relatively buried (ε = 4) or partially solvent-exposed (ε = 30) protein binding pockets based on the fully optimized structures of Na+ and Li+ complexes shown in +-ligating aa ligands may prohibit the incoming Li+ from adopting its favored tetrahedral ligand arrangement, we computed two sets of Na+ → Li+ free energies: those in black characterize cation competition in flexible Na+-sites that permit structural changes upon Li+ binding and those in blue portray the Na+ → Li+ exchange in rigid Na+-sites that prohibit structural reorganization. To model such inflexible binding sites, we fixed the binding cavity's geometry by freezing the protein ligand positions in the fully optimized Na+ complexes and replaced Na+ with Li+, followed by partial optimization, allowing Li+ and the water ligands to find their optimal positions.This was assessed by the free energies for replacing NaGε in +-selective site, whereas a negative value implies a Li+-selective one. Below, we focus on how the Na+ → Li+ free energy changes with varying solvent exposure, rigidity, composition, and net charge of the metal-binding site rather than the absolute free energy. Note that the trends in the free energies computed using the approach herein have been found to be in accord with experimental observations in previous studies.A positive Δ+ is found coordinated to only neutral ligands in several proteins /Asn side chain (modeled by N-methylacetamide) or Ser/Thr side chain (modeled by ethanol) and water molecules. Flexible Na+-binding sites containing many neutral amide residues are prone to Li+ substitution, as evidenced by the favorable Na+ → Li+ free energies in + is a better charge acceptor and forms more favorable interactions with the ligands than the native Na+. Consequently, increasing the number of amides lining the flexible binding pocket enhanced the selectivity for Li+ over Na+: as the number of amide ligands increased from one (Gε (ε = 1–30) became more negative. Such electronic effects favoring Li+ over Na+ are attenuated in solvent-exposed sites, where lithium's higher desolvation penalty outweighs sodium's solvation free energy gain upon Na+ → Li+ substitution (ΔG30 less negative than ΔG4). Hence, buried Na+-sites seem to be more susceptible to Li+ attack than solvent-accessible ones.Since Naproteins , we optifrom one  to two . In contrast to flexible sites, the more crowded a rigid binding site, the more Na+-selective it becomes: as the number of amide ligands in a rigid binding site increased from two to six  increased from –5.6 to 2.8 and 6.4 kcal mol–1, respectively. This is because in a rigid site, Li+ is forced to adopt sodium's longer bond distances, resulting in reduced charge transfer, as compared to flexible sites. Furthermore, the backbone/Asn amides often form hydrogen bonds in the Na+-transporter and receptor structures . They become prone to Li+ substitution if they can undergo structural changes to meet lithium's coordination preferences (G4/ΔG30 in black). Compared to an amide however, the hydroxyl group is a poorer charge donor and its interactions with Li+ are attenuated more than those with Na+, making the Na+-site less susceptible to Li+ attack when one or two amide ligands are replaced by hydroxyl groups (less negative ΔG4/ΔG30 in When we replaced one or two amide ligands in + is found bound to one or at most two acidic ligands in some proteins in +vs. Li+ competition in complexes comprising one or two acetates (modeling Asp–/Glu– carboxylates) and a few neutral ligands. As found above, increasing the number of protein ligands lining a flexible binding pocket favored the cation that is the stronger electron acceptor; i.e., Li+ over Na+: as the number of protein ligands increased from one . As found above, substitution of an amide in G4/ΔG30.Since Nafrom one  to two  may modulate selectivity for Na+ or Li+. Free energy simulations of LeuT transporter also underscore the rigidity of the Na+-binding site, which is enforced by the local structural restraints arising from hydrogen-bonding networks, in determining selectivity for the Na2-site.Rigid Na+ over Li+ in rigid binding sites. Neutral protein ligands that are weak charge-donors such as the Ser/Thr hydroxyl side chain favor Na+ over Li+ in rigid sites, whereas anionic ligands that are strong charge-donors such as the Asp/Glu carboxylates favor the cation with higher charge-accepting ability; i.e., Li+ over Na+.Increasing the number of protein ligands enhances selectivity for Nai.e., Na+ over Li+, whereas a solvent-inaccessible cavity enhances metal–ligand interactions and favors Li+, which can accept more charge and form stronger ligand interactions than Na+.Allowing water into the binding cavity favors the cation with smaller dehydration penalty; +-binding sites containing >3 protein ligands would be well-protected against Li+ attack  and to Li+ +-bound ; 3C32 structure shows Na+ bound to two neutral  and two acidic  aa ligands that altogether form ten hydrogen bonds  that coordinate Na+ adopt similar positions/orientations, but Li+ is closer to Glu96 (Li+–OOE1 = 2.0 Å) at the expense of losing contact with Asp100 (Li+–OOD1 = 3.5 Å). Thus, Li+ does not fit favorably in the rigid cavity and binds with lower affinity than the cognate Na+ to the glutamate receptor 5.+ cannot successfully compete with Na+ bound to two amides and two carboxylates .Interestingly, X-ray structures of the glutamate receptor 5 bound to Naonds see  and S2†.solvent-inaccessible Na+-binding sites lined by only one or two aa residues are susceptible to Li+ attack, regardless of the aa type and relative rigidity of pocket . Such a deeply buried Na+-binding site comprising an Asp– and a Ser is found in two GPCRs; viz., the A2AAR adenosine receptor  and the β1AR adrenergic receptor . Bound Na+ allosterically controls the GPCR activity: It stabilizes the inactive/resting receptor conformation, which cannot transmit signal to the G-protein and associated effectors.+-binding site (by activating transmembrane helix motions) and translocates Na+ to another protein locality,The calculations reveal that + suggests that factors stabilizing the allosteric Na+-binding site would prohibit structural distortions induced by agonist binding, leading to lower cytosolic levels of activated G-proteins, which are hyperactive/overexpressed in bipolar disorder patients. Here, we propose that such a factor is Li+, which can displace Na+ in a buried site , hence it would be interesting to assess if Li+ could displace Na+ in Na-proteins that are ubiquitous in the blood in future studies.Lithium's replacement of Na+ and Na+ for allosteric Na+-binding sites in biologically important neurotransmitter transporters and GPCRs. We show that abiogenic Li+ can target those Na+-binding sites that are flexible with multiple neutral protein ligands or those that are buried containing only one or two aa ligands. By displacing Na+ bound to an Asp– and a Ser in the solvent-inaccessible metal-binding sites of the A2AAR adenosine and the β1AR adrenergic GPCRs, Li+ could stabilize the receptor's inactive state by prohibiting conformational changes to the active state, thus leading to decreased G-protein activity, which are hyperactive in bipolar patients.This is the first study delineating the key factors determining the outcome of the competition between LiThe authors declare no competing financial interest.Supplementary informationClick here for additional data file.