Here, we present new experimental data on the kinetics and regulation of recombination reactions mediated by ϕC31 integrase and its RDF, and use these data as the basis for a mathematical model of the reactions. The model accounts for the unidirectionality of the attP × attB and attL × attR reactions by hypothesizing the formation of structurally distinct, kinetically stable integrase–DNA product complexes, dependent on the presence or absence of RDF. The model accounts for all the available experimental data, and predicts how mutations of the proteins or alterations of reaction conditions might increase the conversion efficiency of recombination.Serine integrases, DNA site-specific recombinases used by bacteriophages for integration and excision of their DNA to and from their host genomes, are increasingly being used as tools for programmed rearrangements of DNA molecules for biotechnology and synthetic biology. A useful feature of serine integrases is the simple regulation and unidirectionality of their reactions. Recombination between the phage  However, the presence of a recombination directionality factor (RDF) protein transforms the activity of integrase so that it promotes L × R recombination, leading to attP and attB products  DNA sites that each bind an integrase dimer. Recombination between the phage-derived ’ Figure . The ‘reproducts –13.The molecular basis for this remarkable directionality is not well understood. It is not accounted for by differences between the free energies of unbound substrate and product DNA molecules , nor is the reaction coupled to any other chemical transformation (such as adenosine triphosphate hydrolysis). It seems therefore that the reactions must be driven by the formation of stable  protein–DNA complexes as end-products. The mode of action of the RDF also remains mysterious. The RDF is known to interact directly with integrase ,13; someMathematical modelling can be used to explore regulatory mechanisms in biological systems , and mayin vitro assays was prepared from the Escherichia coli strain DH5 (F− λ– Δ(lacZYA-argF)U169 recA1 endA1 hsdR17  phoA supE44 thi-1 gyrA96 relA1) using a plasmid miniprep kit (Qiagen) according to the manufacturer's instructions, with one extra wash step with buffer PE. DNA concentrations were determined by measuring the absorbance at 260 nm . Substrate plasmids pPB and pLR  in inverted repeat (separated by 899 bp centre to centre) were constructed in the pMK-RQ plasmid backbone (GeneArt). The invertible DNA segment contains a constitutive promoter, which initiates the transcription of a gfp gene when the segment is in one orientation but not the other. Full sequences of these plasmids are available on request. Oligodeoxynucleotides for assembly of fluorescently labelled linear attB or attP DNA fragments were synthesized by Integrated DNA Technologies (IDT). The sequences of the oligodeoxynucleotides are shown in Supplementary Text S1.1.1. The oligodeoxynucleotides were dissolved at 100 μM in TE0.1 buffer , pH 8.0). Fluorescent labelled oligonucleotides (10 μM 5′-FAM-attP-bot or 5′-Cy5-attB-bot) were mixed with an excess of the corresponding unlabelled oligonucleotide (10.2 μM attP-top or attB-top respectively) in TE0.1 buffer. The mixtures were heated to 87°C for 5 min and cooled down to 25°C over 2 h to anneal the two strands. Recombination converts the 79 bp FAM-labelled attP and 85 bp Cy5-labelled attB to 65 bp FAM-labelled attL and 99 bp Cy5-labelled attR fragments.Substrate plasmid DNA used for R Figure , contain−1 cm−1 for integrase and 43 000 l mol−1 cm−1 for gp3. All protein concentrations given in this paper refer to monomer. Both proteins were diluted in protein dilution buffer containing 25 mM Tris·HCl (pH 7.5), 1 mM DTT, 1 M NaCl and 50% (vol/vol) glycerol, and stored at −20°C.Proteins  were purified as previously described ,12,18. PattP and attB (10 nM of each) were added instead of plasmid.Aliquots of ϕC31 integrase or integrase premixed with RDF were prepared in protein dilution buffer at 10 times their desired final concentrations. Reactions (20 μl for the 3 h end-point measurements or 130 μl for the time courses) were set up by pre-incubating 10 nM plasmid substrate in a buffer containing 25 mM Tris·HCl pH 7.5, 2.5 mM spermidine, 25 mM NaCl and 50 μg/ml bovine serum albumin (BSA) at 30°C for 10 min. Recombination was then started by adding protein solution , and reactions were incubated at 30°C. For the time courses, 10 μl aliquots were taken and stopped at each time point. Other reactions were for 3 h at 30°C. For all reactions, recombination was stopped by heating the samples at 80°C for 10 min. For reactions of linear DNA substrates, annealed oligonucleotide E. coli cells and counting the proportion of transformant colonies that expressed GFP (data not shown). All experiments were carried out in triplicate. Average values and standard deviations are shown on all experimental figures.Reactions of plasmid substrates were analysed by restriction enzyme digestion followed by gel electrophoresis. After digestion with restriction enzymes EcoRI and KpnI at 37°C for 3 h, 5 μl of loading buffer (25 mM Tris·HCl (pH 8.2), 20% (w/v) Ficoll, 0.5% sodium dodecyl sulphate, 5 mg/ml protease K, 0.25 mg/ml bromophenol blue) was added to each sample, and the mixture was incubated at 37°C for 30 min prior to loading onto a 1.2% (wt/vol) agarose gel (24 × 16 × 0.6 cm) in Tris-Acetate EDTA (TAE) buffer . Gels were run for 16 h at 0.65 V/cm, stained extensively (>30 min) with ethidium bromide (0.5 μg/ml), destained in TAE buffer for 10 min and imaged using a Bio-Rad GelDoc UV Transilluminator. The intensities of DNA bands on the gel were quantitated using the volume analysis tool of Quantity One software (Biorad) using background rectangle subtraction, and the proportions of recombinant products were determined after correcting the intensity values for fragment size. The accuracy of quantitation by this method was confirmed by introducing product DNA into chemically competent Recombination of linear substrates was assayed by running 8% polyacrylamide gels (37.5:1 acrylamide:bisacrylamide) in Tris/Borate/EDTA (TBE) buffer . Gels were scanned using a GE Healthcare Typhoon FLA9500 laser scanner in fluorescence mode set to detect FAM (473 nm laser and 510 nm long pass LPB filter) and Cy5 (635 nm laser and 665 nm long pass LPR filter). The fluorescent intensities of linear DNA bands on the gel were quantitated using the volume analysis tool of Quantity One software (Biorad).attP × attB and attL × attR reactions respectively, and the abbreviations pPB and pLR to refer to the corresponding plasmid substrates. We also developed a version of Model 1 for intermolecular recombination of linear DNA substrates  described in the relevant Results section. ODEs of all models and parameter values are presented in Supplementary Text S1.2 and Supplementary Tables S1–3.Our modelling uses ordinary differential equations (ODEs) to describe the kinetics of the reaction systems . The modKb1, Kb2, Kb3, Kb4; pairing of two DNA-bound integrase dimers or integrase–RDF complexes to form a synapse (synapsis), which are called ‘s’ steps, with dimensionless (for models of plasmid recombination) equilibrium constants Ks1, Ks2, Ks3, Ks4; and recombination steps called ‘r’ with the equilibrium constants called Kr1, Kr2. Additionally, Model 1 has two steps (‘mod’ and ‘modr’) corresponding to conformational changes (modification) of synaptic complexes, with equilibrium constants Kmod, Kmodr. The motivation for including these modification steps is described in the Results section. Interactions between integrase and RDF in solution were modelled with dissociation constants Kii for integrase dimerization and Kir for binding of integrase to RDF . In addition, the observed inhibition of the P × B reaction at high integrase concentrations was modelled through the formation of non-functional multimeric complexes of integrase with PB substrate . The 25 Model 1 parameters were fitted to our experimental data (presented in Supplementary Table S4), which contains 92 data points including time courses and 3-h end-point measurements under different integrase and RDF concentrations. Different parameters are constrained by different aspects of the data. Some representative examples of the effects of parameters on the reaction outcomes are shown in Supplementary Figure S3. Supplementary Text S1.2.1 gives further details on the relationships between the parameters and the modelled outcomes. Briefly, the affinities of binding of integrase dimers and integrase–RDF complexes to the DNA are constrained by the dependence of reactions on integrase and RDF concentrations , using concentrations of 10 nM for DNA-containing species and 200 nM for unbound protein complexes, where Γ is the mass action ratio and Keq is the equilibrium constant of the corresponding reaction.All reaction steps of our models are assumed to be reversible and described by first or second order kinetics. Therefore, each reaction step is characterized by two parameters; the rate constants of forward and reverse reactions, with the equilibrium constant for each step defined as the ratio of forward to reverse rate constants. Equilibrium constants are named according to the steps that they represent Figure : integraing gels ). The paing gels ,21. The A 2-fold (100%) change in any parameter of Model 1 causes <33% change in the maximal yields of reaction products . The model is thus very robust to parameter perturbations.https://github.com/QTB-HHU/integraseModel.The system of ODEs was solved using MATLAB, integrated with the stiff solver ode15s . The MATLAB code of the model is provided in Supplementary Text S1.4 and is freely available at attP to attB (or attL to attR) sites have been adjusted in some experiments to maximize recombination of one of the substrates (in vitro. The attP × attB (pPB) and attL × attR (pLR) plasmid substrates both have two att sites in inverted repeat and are identical except for the att sites and the orientation of the DNA segment between the two sites (the pLR substrate is the recombination product of the pPB substrate and vice versa) , avoids the potential bias due to entropy changes that might be introduced in reactions that change DNA geometry . For comparison, we also conducted experiments to assay intermolecular recombination between attB and attP sites located on separate linear (oligonucleotide) DNA molecules.Published data on the kinetics of ϕC31 integrase-mediated reactions have been obtained under a variety of experimental conditions, and using different kinds of DNA substrates. Intramolecular reactions between sites on supercoiled plasmids, intermolecular reactions between two linear molecules and intermolecular reactions between linear and circular plasmid molecules have been reported ,18,19,22bstrates ,22. It i) Figure . Use of attP × attB recombination (substrate pPB) reached 80% at 200–400 nM integrase, and decreased slightly at higher integrase concentrations. For maximal recombination, an excess of integrase (or integrase and RDF) over substrate binding sites was required . Excess integrase might be required because non-specific binding to DNA reduces the amount of integrase available to bind at the att sites. There was no recombination at all between attL and attR (substrate pLR) after 3 h of incubation with integrase in the absence of RDF, in agreement with existing in vitro data , one or both of these reactions must not be at equilibrium after 3 h. The possible reasons for this disequilibrium will be discussed below.First, we measured the effects of varying integrase and RDF concentrations on the extent of recombination after 3 h Figure  and B. Ttro data . These rThe presence of RDF enables recombination of the pLR substrate Figure  and 4B aWe then measured the kinetics of recombination in time course reactions with 400 nM ϕC31 integrase and 10 nM of plasmid substrate, in the absence (for pPB reactions) or presence (for pLR reactions) of 800 nM of RDF. In both cases, the recombinant product level quickly reached a maximum Figure , but notatt sites in a single plasmid. Our experimental analysis confirms that this is a good approximation; the amount of plasmid dimer (the most abundant intermolecular product) is typically <5% of total product (data not shown). (ii) Integrase monomers interact to form dimers in solution, which then bind to the DNA. This assumption is supported by published data . The attP × attB reaction in the absence of RDF (P × B (−RDF)) thus starts with binding of two dimers of integrase to the att sites in the pPB plasmid substrate . These two steps are modelled to be energetically favourable  to match the experimental data. The simplest version . It is known that strand exchange is a complex process involving strand cleavages, subunit rotation and strand re-ligations, steps that are likely to be accompanied by protein conformational changes  then dissociates (‘desynapsis’) to form the pLR product plasmid with two bound integrase dimers (step s2 (Model 0), forming LR–int). Desynapsis in step s2 is assumed in Model 0 to be favourable  then takes place . Similarly to the P × B (−RDF) reaction, the binding of integrase–RDF complexes to pLR and formation of the synapse (LR–int–rdfs) are assumed to be energetically favourable steps . The synapse PB–int–rdfs1 then dissociates , forming PB–int–rdf). Finally, dissociation of the two int2–rdf2 complexes from the recombinant att sites (step b4) would generate unbound pPB. However, as for the P × B (−RDF) reaction described above, protein-bound pPB is expected to be energetically favoured.Model 0 also describes the L × R reaction in the presence of RDF (L × R (+RDF)). The current structure-based hypothesis  proposess Figure . Next, tModel 0 also includes unproductive pLR complexes formed with two integrase dimers and one, two, or three RDF monomers . The model assumes that only complexes containing four molecules of RDF are productive. The effects of insufficient RDF might be at pre- or post-synaptic steps. The formation of unproductive complexes reduces the amounts of reaction products in the L × R (+RDF) reaction when the RDF:integrase ratio is reduced below 1:1, in agreement with the data Figures  and 4B. Our models ignore possible effects on the energetics of the reaction due to topological changes in the plasmid DNA during recombination. Current data suggest that the products of an inversion reaction of a supercoiled plasmid substrate are likely to comprise a complex mixture of topologies including a large proportion of knotted molecules, and it is also likely that these ‘knotting’ topological changes along with associated changes in DNA linking number are energetically favourable overall ,18,27. HattP, attB, attL and attR sites, which are all quite similar . The conservation of energy in a chain of reversible reactions in a closed cycle  can be formally described by the so-called Wegscheider's condition , which r similar .s1 synapse from LR–int), would require an identical reduction of the rate constant for the same step in the forward (pPB to pLR) direction to keep the equilibrium constant for this step unchanged. These changes would reduce the rate at which the reaction approaches equilibrium in both directions, in disagreement with our data.A second problem is that in Model 0, all the reactions quickly approach equilibrium in both directions, yielding similar ratios of pPB to pLR in 3 h, regardless of whether the reaction starts from pPB or pLR, in contradiction to our experimental data Figure  and B. Ts2 and PB–int–rdfs2; Figure s2 is formed from LR–ints1 , in better agreement with published data  reaction, with its unfavourable desynapsis in step s4  are slow . This allowed us to account for the observed difference in pLR:pPB ratio at the end of reactions started from pPB or pLR  reaction, and thus a very slow approach to equilibrium. On a short timescale, the L × R (−RDF) reaction proceeds only as far as binding of the pLR substrate by integrase dimers in Model 1, as discussed further below. Similarly, for the L × R (+RDF) reaction, we hypothesize that the forward and reverse rate constants of step s4 (desynapsis/synapsis of the stable synaptic complex PB–int–rdfs2 to PB–int–rdf) are low. We can thus explain the observed difference in the pLR:pPB ratio at the end of reactions started from pLR or pPB in the presence of RDF, by the very slow approach of the P × B (+RDF) reaction to equilibrium  and P × B (+RDF)) reach equilibrium too quickly in Model 0 Figure  and B, ww Figure . For theR Figure , as a com Figure .att sites, so that an att site may be bound by an integrase tetramer and thus become incompetent for synapsis   .Model 1 accurately matches our data for the kinetics of the P × B (−RDF) and L × R (+RDF) reactions on plasmid substrates Figure . The mods1 and LR–ints2 synaptic complexes represent the two major pLR fractions at equilibrium ) should thus be effectively irreversible under natural in vivo conditions, where events such as chromosome replication and cell division would typically occur on a faster timescale. Sequestration of the free LR product into a non-productive complex with integrase (LR–int) might be a viral strategy to avoid spontaneous provirus excision in the absence of RDF.Model 1 predicts that the P × B (−RDF) reaction rapidly approaches equilibrium, reaching 68% of pLR recombinant product in 30 min and 75% of pLR at final equilibrium, at an integrase concentration of 400 nM Figure . The LR– Figures  and 6A.  Figures , because Figures , which is2, which can then equilibrate with PB–int–rdfs1 and LR–int–rdfs ; theatt sites within a single supercoiled plasmid molecule), whereas the natural P × B reaction is intermolecular , and intermolecular reactions (both P × B and L × R) are required for many proposed applications. Therefore we measured the experimental kinetics of the P × B (−RDF) reaction with linear DNA substrates. Recombination between linear (oligonucleotide) attP and attB substrates is slower than intramolecular recombination of a plasmid substrate  compared to 10 nM plasmid substrate under the same conditions  to product can be enhanced by increasing concentration of the second substrate  reproduces the observed behaviour of the ϕC31 site-specific recombination system and provides a putative molecular explanation for directionality and the action of RDF.et al. suggest that the observed incomplete conversion of substrate to product in vitro is due to irreversible inactivation of integrase during the course of the reaction  together  attP and (shorter) attB sites, but are favourable in dimers bound to the intermediate-length attL and attR sites . These tR sites –16.et al. (s1) dissociates to give the pLR product plasmid bound by two integrase dimers . Thet Figure , allowintR sites ,23.s2 is not a synapse, but is pLR plasmid bound to two separate integrase dimers, in a conformation different from the LR–int complex formed after binding of integrase to pLR. The transition between these two configurations of LR–integrase complexes should be very slow accordingly to our model. The model and the existing data do not allow discrimination between these two possibilities.Stable integrase–product DNA synaptic complexes have not yet been observed experimentally in biochemical assays , though s2 and the non-synapsed pLR plasmid bound by integrase dimers (LR–int) is very slow. This assumption allows Model 1 to account for the unidirectionality of P × B recombination in the absence of RDF  is promoted. In our Model 1, this corresponds to rapid synapsis of attL and attR sites bound by integrase–RDF complexes  which is the normal end-point of the reaction and slow interconversion of this synaptic complex with the product plasmid bound by two separate integrase dimer–RDF complexes .In the presence of RDF, integrase dimers bound to d et al. , RDF migs Figure , step s3att sites, or by changing the reaction conditions or substrate structure) could increase the conversion efficiency of the reactions, as might be desirable for many applications in biotechnology. For pPB recombination, a 2-fold increase of either of the equilibrium constants Kr1 or Kmod (with a compensating change in Ks2 to fulfil Wegscheider's condition) increases the pLR product yield at equilibrium from 76 to 86% . Likewise, a 2-fold increase of Kr2 or Kmodr for the pLR (+RDF) reaction increases the pPB product yield from 67 to 80%. Differences in the observed conversion efficiencies of natural serine integrases . Our Model 1 accounts for this incomplete conversion as being primarily due to fast equilibration of synapsed forms of the substrate and product species . The model therefore suggests that alterations to the equilibrium constants of these steps (by mutation of the proteins or tegrases  might si


Alzheimer's disease (AD) is characterized by two molecular pathologies: cerebral β-amyloidosis in the form of β-amyloid (Aβ) plaques and tauopathy in the form of neurofibrillary tangles, neuritic plaques, and neuropil threads. Until recently, only Aβ could be studied in humans using positron emission tomography (PET) imaging owing to a lack of tau PET imaging agents. Clinical pathological studies have linked tau pathology closely to the onset and progression of cognitive symptoms in patients with AD. We report PET imaging of tau and Aβ in a cohort of cognitively normal older adults and those with mild AD. Multivariate analyses identified unique disease-related stereotypical spatial patterns (topographies) for deposition of tau and Aβ. These PET imaging tau and Aβ topographies were spatially distinct but correlated with disease progression. Cerebrospinal fluid measures of tau, often used to stage preclinical AD, correlated with tau deposition in the temporal lobe. Tau deposition in the temporal lobe more closely tracked dementia status and was a better predictor of cognitive performance than Ab deposition in any region of the brain. These data support models of AD where tau pathology closely tracks changes in brain function that are responsible for the onset of early symptoms in AD. Alzheimer's disease (AD) is characterized neuropathologically by the presence of β-amyloid (Aβ) plaques and tau immunoreactive neuritic plaques, neurofibrillary tangles, and neuropil threads in the cerebral cortex . AutopsyExtant models of AD pathophysiology, such as the amyloid cascade hypothesis, establish a temporal ordering of markers, with the emergence of AD-related tauopathy occurring downstream to the accrual of Aβ pathology –11. ConsThe use of both tau and Aβ imaging allows for the attribution of AD-related cognitive dysfunction to specific pathological processes and locations within the brain. Further, the spatial relationships between tau pathology and other markers of AD have not been established. Here, we examine how tau imaging topographies relate to clinical status, Aβ imaging, CSF measures of AD pathology, and neuropsychological performance. Tau and Aβ represent distinct pathological processes, but they are strongly related in the context of AD. Given that PET- and CSF-based techniques may measure similar processes , PET andData from 46 individuals were analyzed . Demographics of each subset of the cohort are provided in n = 36) participants showed minimal tau tracer uptake throughout the brain with the exception of the basal ganglia. Cognitively impaired  participants exhibited markedly increased tau tracer uptake in the temporal lobes and throughout the cerebral cortex. Aβ imaging showed the known difference in measured Aβ deposition between cognitively normal and cognitively impaired groups (Mean (across participants) tau and Aβ SUVR imaging data are presented in d groups .n = 46) by ROI matrices (M = 42) separately and subjected to SVD. PET imaging of tau and Aβ was best described as a combination of two component topographies  it accounted for the correlation structure of the data across ROIs and (ii) reduced the number of statistical comparisons. Singular value decomposition (SVD) identified a small number of latent component topographies whose representation in single subjects explained a majority of the variance present in the data. In each participant, tau and Aβ pathology burden measured by PET imaging was assessed in 42 ROIs; many ROIs were highly correlated with other ROIs and could be more concisely summarized as topographies or combinations of ROIs. The PET imaging tau and Aβ data were arranged into participants (graphies . Both PEP < 0.001) but were not significantly modified by age or APOE status (P = 0.04) and moderately associated with increasing age (P = 0.08). In both the tau and Aβ data, there were statistically significant differences between the CDR0 and CDR>0 groups; however, there was a significant overlap particularly in the Aβ data  with age, CDR, and presence of an APOE ε4 allele as factors . The twoE status . The fir Aβ data . The sepF values ; these Fr PET Aβ . Thus, aInspection of r = 0.29, P = 0.062), which reflects the known divergence in the pathological topographies , which reflects the strong relationship between tau and Aβ when the appropriate topographies are considered. The PET tau topography was most heavily localized in the medial temporal lobe, parietal cortex, and precuneus and was most highly correlated with PET Aβ topography in the frontal and parietal regions.The previous analysis described the relationship between PET tau and PET Aβ topographies and disease stage. Next, the relationship between the PET tau and Aβ topographies was compared directly using canonical correlation. Canonical correlation is a multivariate generalization of the more familiar bivariate correlation. Canonical correlation identified both a PET tau and PET Aβ topography such that the data, when projected onto these weighted topographies, were maximally correlated. Thus, canonical correlation identified the topography of each pathological species that was mostly related to the other species. The present PET tau and PET Aβ topographies were represented by a single canonical correlation that explained 75% of the covariance in the data . The weigraphies . The PET42 were calculated  and CSF p-tau  but did not significantly correlate with CSF Aβ42 ; mean PET Aβ negatively correlated with CSF Aβ42  but also positively correlated with CSF tau  and p-tau . The negative correlations were due to CSF Aβ42 being reduced, not increased, during AD pathogenesis as more Aβ becomes deposited in the brain.PET tau and Aβ imaging techniques and CSF-based techniques may measure correlated pathological processes . Therefolculated . As pred42 (separately) as an outcome and regional PET tau and Aβ SUVR (together) as predictors were fit to determine the relationship between CSF and regional PET measures. PET tau and PET Aβ topographies were considered together in the model, allowing for the data to determine the modality that best predicted the variable of interest. The critical feature of penalized regression is that the solution is sparse , and only the most predictive ROIs receive nonzero regression β weights. The relationship between PET tau and Aβ topographies and CSF tau was examined first. The penalized model significantly fit the data . The combination of PET tau and Aβ topographies that best correlated with CSF tau is shown in the top row of tau| = 2.93) and PET Aβ (Σ|βAβ| = 2.54), as measured by the sum of the absolute value of the regression β values corresponding to PET tau and Aβ ROIs, respectively. This equal weighting was consistent with tau pathology being dependent on initial Aβ accumulation. Predictive PET tau regions included entorhinal and temporal cortex and the cuneus. The PET Aβ contribution was derived from the temporal and frontal regions. Similar topographies of predictive PET ROIs were found when predicting CSF p-tau; the overall correlation between the CSF tau and CSF p-tau predictive topographies was high .Penalized regression models –27 with 42 as the predicted outcome variable. The penalized model significantly fit the data . The joint topographies of PET tau and Aβ that best correlated with CSF Aβ42 are shown in the bottom row of Aβ| = 0.46) compared to PET tau (Σ|βtau| = 0.26). This suggested that PET tau has relatively less predictive information relating to CSF Aβ42. The contributing topography from PET tau was primarily loaded in the superior portions of the cortex. The PET Aβ topography was primarily composed of frontal and parietal regions. The highly significant predictive power of both models suggested that there was a strong relationship between CSF and PET measures of tau and Aβ pathology.Next, the same relationship was investigated, but with CSF AβZ = 2.42, P = 0.0078), semantic , visuospatial , and the global score  were significantly predicted by penalized regression after correction for multiple comparisons, whereas the working memory composite was not significantly fit by the model. The combined PET tau and Aβ topographies that best predicted each cognitive composite are shown in To examine the relationship between pathology and cognitive function, the relationship between PET tau and Aβ topographies and neuropsychological performance composite scores was compared using the same penalized regression model as described above. Each participant underwent neuropsychological testing that was summarized into four composite scores summarizing the domains of episodic, semantic, working memory, and visuospatial processing and an additional overall  measure. Of these, episodic . Similarly, the topographies predictive of semantic memory performance were not significantly correlated . However, the topographies predicting visuospatial performance  and global cognitive performance  were correlated . These data suggest that the relationship between pathology and neuropsychological performance varies over the course of the disease.We further investigated the correlation between PET tau and Aβ topography and neuropsychological composite scores in the CDR0 group only. In the domains that were significantly associated with PET tau and Aβ topographies in the full group, we refitted the models in the CDR0 group only and calculated the correlation between the resulting predictive topographies. The topography that was predictive of episodic memory scores in the full group was not correlated with the predictive topography in the CDR0 group  and have identified PET tau topographies consistent with studies in postmortem brain tissue  and neuropsychological assessment (n = 40). All participants or their designees provided written informed consent before participating in this study that was consistent with the regulations of the Washington University in St. Louis School of Medicine Institutional Review Board.Forty-six participants were recruited from ongoing studies of memory and aging at the Knight Alzheimer's Disease Research Center at Washington University in St. Louis. Participants underwent cognitive assessment with the CDR scale , and MRIA comprehensive neuropsychological battery was administered to all participants, typically within 2 weeks of the annual clinical assessment. Standardized test scores were averaged to form four composites [see  for detaMRI and florbetapir data were acquired on a Biograph mMR scanner  in a single session. T807 data were acquired in a separate session on a Biograph 40 PET/CT (computed tomography) scanner. Participants received a single intravenous bolus injection of 10 mCi of florbetapir and a single intravenous bolus injection of 6.5 to 10 mCi of T807.http://github.com/ysu001/PUP). FreeSurfer segmentation (http://freesurfer.net/) was used as the basis of the quantitative analysis to obtain regional SUVR with cerebellar gray matter as the reference region. Partial volume correction was also performed using a regional spread function technique ].CSF (20 to 30 ml) was collected after overnight fasting as described previously , 49. TotPET SUVR, for both T807 and florbetapir, can be measured at the resolution of voxels, structurally or functionally defined sets of voxels, or the entire brain. This gives rise to data sets that appear to be high-dimensional  but can be represented by lower-dimensional projections. These bases can be identified using SVD or related procedures . In PET data, these composite factors represent topographies , and the contribution of a given topography to an individual participant's data can be estimated. SVD was performed on the regional SUVR values (as variables) across participants (as samples) for tau and Aβ separately. The number of significant factors was determined by a permutation test.42 or neuropsychological Z scores (as outcome variables) and PET tau and Aβ topographies (as predictors). The penalty in penalized regression is enforced against the estimated regression βs  results related to SVD topographies.Table S2. Mean PET and CSF correlation matrix.Table S3. Regional regression β values.Table S4. Comparison of PET tau SVD results.Table S5. Leave-one-out analysis results.


Here we observe that the interfacial coupling between the 3d antiferromagnetic insulator SrMnO3 and the 5d paramagnetic metal SrIrO3 is enormously strong, yielding an anomalous Hall response as the result of charge transfer driven interfacial ferromagnetism. These findings show that low dimensional spin–orbit entangled 3d–5d interfaces provide an avenue to uncover technologically relevant physical phenomena unattainable in bulk materials.Strong Coulomb repulsion and spin–orbit coupling are known to give rise to exotic physical phenomena in transition metal oxides. Initial attempts to investigate systems, where both of these fundamental interactions are comparably strong, such as 3 Whilst superlattices containing thin films of 5d transition metal oxides are expected to yield strong interfacial coupling, only weak effects have been observed. Here, the authors report strong coupling between 3d SrMnO3 and 5d SrIrO3 due to the interplay of strong Coulomb and spin orbit interactions. Even though there are a few examples of successful synthesis of 3d–5d superlattices141516Jeff=1/2 that is induced by strong spin–orbit coupling (SOC), there has been enormous interest in many Ir-based 5d TMOs181920ξ) with an energy scale comparable to the on-site Coulomb interaction (U)24d systems. However, these novel ground states are yet to be experimentally confirmed.The strong interplay among charge, spin, orbital and lattice order parameters in transition metal oxides (TMOs) is known to produce exotic physical phenomena58910113 (SMO), a 3d TMO with weak ξ (0.01–0.1 eV) strong U (5–7 eV), and the paramagnetic metal SrIrO3 (SIO), a 5d TMO with strong ξ (0.1–1 eV) and modest U (1–3 eV)d–5d interfaces where collectively both U and ξ are stronger than in either parent compound. Interestingly, we find that our [(SMO)m/(SIO)n]z (MmIn) heterostructures, where m and n are, respectively, the thicknesses of SMO and SIO in unit cells, display exceptionally strong interfacial coupling between the two constituent materials, yielding a ferromagnetic ground state. Such emerging interfacial magnetism in turn results in a strong anomalous Hall effect (AHE). As the emergence of ferromagnetism and the AHE are completely absent from either parent compound, this discovery provides the first experimental evidence of strong coupling at the interface of 3d and 5d materials.To narrow this gap between experimental and theoretical efforts, we have synthesized atomic-scale heterostructures by incorporating the antiferromagnetic insulator SrMnOH) dependence of symmetric samples (m=n) is presented in M) of M1I1 is significantly larger than twice that of M2I2 along with M4I4 having M≈0 implies that interfacial diffusion is not responsible for the magnetic properties here and the mechanism driving this induced interfacial magnetism must have a characteristic length scale of just a few unit cells. The temperature-dependent nature of the magnetization of these samples is shown in m>3 showed no magnetic order, while below this limit, the magnitude increased with decreasing m. The Curie temperature (Tc) is shown in the inset where M1I1 has the largest Tc∼190 K. Note that M1I1 has a second anomaly at ∼120 K for H⊥c that is likely associated with its electronic properties as discussed below. The magnetic anisotropy of a second M1I1 sample is presented in Tc are independent of the direction of H, both the coercive field and remnant magnetization are roughly an order of magnitude larger when H is parallel to the c axis (out of plane). This result implies that the c axis is the magnetic easy axis.The first indication of such unique behaviour is the onset of magnetism in atomically thin superlattices. The macroscopic magnetic properties were measured with a superconducting quantum interference device (SQUID) and are shown in L3 and L2 edges of both Mn and Ir -doped SMO (SIO) layers. The average oxidation states are estimated from the peak shifts and are presented in the inset of 1I1 clearly has the largest deviation from the nominal value with a charge transfer of ∼0.5 electron/hole per perovskite unit cell. Although in absolute units these estimates of the oxidation state have a relatively large uncertainty, it is important to note that their relative uncertainties are significantly smaller than the data points. The XMCD spectra of the Mn L3 edge show that M1I1 has a large negative response, which indicates that the magnetic moment of the Mn ions (MMn) orders parallel to H. As m increases, the Mn XMCD decreases. Despite the consistency between SQUID and Mn XMCD measurements, there are surprisingly finite XMCD peaks near the Ir L edges. This XMCD result implies that there is a net magnetic moment of Ir (MIr) due to the onset of ferromagnetism or canted antiferromagnetism. The observation of net ferromagnetic order of Ir ions is quite surprising since Ir4+ and Ir5+ tend to favour antiferromagnetic2628S) and orbital (L) contributions of MIr and found them to be 0.013 μB and 0.057 μB, respectively, for M1I1, whereas for MMn, S and L are 0.9 μB and 0.3 μB, respectively. Combining these results, we determine the total magnetization (M=L+2S) in each material to be MIr=–0.08 μB/Ir and MMn=2.1 μB/Mn, which are in good agreement with SQUID data. Thus, we conclude that MMn is mostly driven by spin, while MIr has predominately orbital contributions due to strong SOCL3/IL2) of Mn in SMO is ∼2 and systematically increases with decreasing m. Although this qualitative behaviour can be explained by the reduction of the Mn oxidation state, it is worth noting that a BR>2 is often attributed to the presence of spin–orbit interactionsTo fully understand the magnetism of these superlattices, it is necessary to identify the relative contribution of Mn and Ir ions to the overall magnetic moment. Both X-ray absorption spectroscopy (XAS) and X-ray magnetic circular dichroism (XMCD) spectra provide information rich with elemental-specific contributions regarding both the electronic and magnetic structures. Thus, we collected XAS and XMCD spectra near the n and Ir  to underm≤3) are magnetic and all superlattice Bragg peak positions of these samples lie at wavevector transfer (q) values unobtainable with reasonable measurement parameters. This challenge was overcome by synthesizing an asymmetric M1I10 sample, which has a larger superlattice period and an appreciable magnetic response  profiles obtained from spin-dependent PNR measurements and X-ray SLD profile from X-ray reflectometry are shown in MMn is much larger than MIr, which is consistent with XMCD measurements. However, conversely, our PNR indicates that MIr aligns parallel to the applied magnetic field in M1I10, whereas XMCD has revealed that it aligns antiparallel for M1I1. This discrepancy suggests that there is a critical SIO thickness, in which the Ir moments realign. Confidence in this interpretation of non-zero MIr arises from the fact that if the MIr is forced to zero n (n≤3)d and 5d TMOs.The microscopic origin of the magnetism was further investigated by polarized neutron reflectometry (PNR), which is a sensitive probe of spin asymmetry. This technique provides a detailed look at the magnetism of thin films and heterostructures as a function of depth. However, PNR of our symmetric magnetic superlattices is a formidable task since only short-period superlattices  is shown in RS (300 K)∼1 MΩ) and SIO is semimetallic. The fact that all samples are roughly 50 nm thick and the resistance of M12I12 is approximately double that of SIO implies that the SIO layers in long-period superlattices (m≳12) dominate the overall electronic conduction. However, when the layer thicknesses are intermediately thick (3≤m≤6), the heterostructures have significantly enhanced metallicity with a weak upturn below 50 K, which is most probably due to weak localization. In this intermediate thickness region, there is minimal charge transfer, which implies that the magnitude of electron (hole) doping of the SMO (SIO) layers is quite small. Since bulk SMO is known to be insensitive to small concentrations of electron dopingm<3), the resistance increases as shown in 1I1, which displays a semimetallic behaviour with a local maximum at ∼120 K. Comparing this with comparably doped bulk Lax1−SrxMnO3 (x=0.55)1I1 superlattice. Thus, the resistivity in small-period superlattices is explained by the large electron doping concentration in atomically thin SMO layers, resulting in a finite electrical conductivity accompanied by the atomically thin SIO layers having reduced conductivity due to reduced dimensionality351I1 is due to the onset of conductivity in the SMO layers.The electronic properties of the symmetric samples were investigated via DC transport measurements, and the temperature-dependent sheet resistance , a butterfly hysteresis loop appears at small H that is coupled with the coercive field as comparatively shown in Rxy) of our superlattices lead to an unprecedented observation. Consider the temperature-dependent Hall resistance of the M1I1 superlattice shown in Tc, the Hall response is linear with a negative slope, indicating n-type carriers. Below Tc, a nonlinear AHE appears, opening a large hysteresis loop at low temperatures with a shape and coercive field practically identical to M(H) sweeps obtained from SQUID measurements (The intriguing magnetic properties of these superlattices were further investigated via magnetoresistance (MR) and Hall measurements presented in 1 sample  has a neurements . In addiσxy) and longitudinal conductivity (σxx) of σxy∝σxx1.6; (ii) the super-clean metal limit where skew scattering off extrinsic defects leads to a scaling relationship of σxy∝σxx; and (iii) the moderately dirty metal region where intrinsic scattering leads to σxy being approximately independent of σxx  than ϕ=1.6. Recall that, although the AHE resides in the SMO layers, the XMCD hinted that SOC substantially influences the SMO layers. Since the magnitudes of σxx that separate the three regions described above depend inversely on ξ, for 5d materials, the moderately dirty limit is roughly 45≤σxx≤6,800 Ω−1 cm−1. This remarkable agreement with our experimental results strongly suggests that SOC is instrumental in defining the novel magnetic and electronic ground states of these 3d–5d TMO heterostructures, and that they are near the moderately dirty limit that has a characteristic dissipationless AHE currentσxy observed here is significantly lower than the theoretical intrinsic scattering limit of ∼900 Ω−1 cm−1 proposed by the Thouless–Kohmoto–Nightingale–Nijs formulismxy should scale with ξ. We attribute this discrepancy to the fact that, although the AHE resides in the SMO layers, the SIO layers still conduct appreciably well and serve as a resistive short of the voltage leads during the Hall measurements, which greatly reduce the measured values of σxy.Since the dominant magnetic ion is Mn and the AHE is driven by magnetism, it is logical to assume the majority of AHE resides within the SMO layers. Recent advances in understanding the AHE separates such materials into three categories: (i) the dirty metal limit where intrinsic and side jump scattering leads to a scaling relationship between the transverse conductivity m/(SrIrO3)n]z were synthesized by pulsed laser epitaxy on atomically flat TiO2-terminated (100) SrTiO3 substrates utilizing a KrF eximer laser (λ=248 nm) with laser fluence, substrate temperature and oxygen partial pressure of 1.0 J cm−2, 700 °C and 100 mtorr, respectively. The crystal structure, orientation, phase purity and crystallinity of these superlattices were determined by X-ray diffraction and reflectivity measurements.The superlattice samples of [The macroscopic magnetic properties were characterized with a 7 T Quantum Design MPMS3. The XAS and XMCD spectra near the Mn and Ir The data that support the findings of this study are available from the corresponding author on request.How to cite this article: Nichols, J. et al. Emerging magnetism and anomalous Hall effect in iridate–manganite heterostructures. Nat. Commun. 7:12721 doi: 10.1038/ncomms12721 (2016).Supplementary Figure 1-4, Supplementary Note 1 and Supplementary References


HH) antibodies specific for the α-synuclein protein (whose aggregation is associated with Parkinson’s disease) with the greatest gains in affinity (>5-fold) have several (four to six) CDR mutations. This finding highlights the importance of sampling combinations of CDR mutations during the first step of affinity maturation to maximize the efficiency of the process. Interestingly, we find that some natural diversity mutations simultaneously enhance all three key antibody properties  while other mutations enhance some of these properties  and display trade-offs in others . Computational modeling reveals that improvements in affinity are generally not due to direct interactions involving CDR mutations but rather due to indirect effects that enhance existing interactions and/or promote new interactions between the antigen and wild-type CDR residues. We expect that natural diversity mutagenesis will be useful for efficient affinity maturation of a wide range of antibody fragments and full-length antibodies.The identification of mutations that enhance antibody affinity while maintaining high antibody specificity and stability is a time-consuming and laborious process. Here, we report an efficient methodology for systematically and rapidly enhancing the affinity of antibody variable domains while maximizing specificity and stability using novel synthetic antibody libraries. Our approach first uses computational and experimental alanine scanning mutagenesis to identify sites in the complementarity-determining regions (CDRs) that are permissive to mutagenesis while maintaining antigen binding. Next, we mutagenize the most permissive CDR positions using degenerate codons to encode wild-type residues and a small number of the most frequently occurring residues at each CDR position based on natural antibody diversity. This mutagenesis approach results in antibody libraries with variants that have a wide range of numbers of CDR mutations, including antibody domains with single mutations and others with tens of mutations. Finally, we sort the modest size libraries (~10 million variants) displayed on the surface of yeast to identify CDR mutations with the greatest increases in affinity. Importantly, we find that single-domain (V Moreover, improvements in antibody affinity are generally expected to enhance the performance of diagnostic antibodies due to improved specificity at reduced antibody concentrations. Methods such as phage, yeast surface and ribosome display are commonly used for in vitro affinity maturation because of their many attractive properties , single mutations typically do not result in large gains in affinity  domain—originally referred to as NbSyn2 and herein referred to as N2—was previously isolated from an immune library. We selected this antibody domain for further optimization because its crystal structure is available in complex with antigen at high resolution  and/or multichain (Fab or IgG) antibodies, it has intermediate affinity (DK of 58 ± 9 nM) that can be further increased, and it has relatively high stability (apparent melting temperature of 68 ± 0.3°C). We posit that efficient affinity maturation of antibody variable domains such as N2 can be accomplished in three steps: (i) identification of the most permissive sites in the CDRs that can be mutated without large (negative) impacts on affinity using alanine scanning mutagenesis; (ii) sampling of a small number of mutations at each permissive CDR site that correspond to either the wild-type residue or residues most commonly observed in natural antibodies at each CDR site; and (iii) screening of all possible combinations of single and multiple natural diversity antibody mutations in a single library. Here, we test this methodology by identifying the most permissive CDR sites in N2 and use these findings to generate a single library that is based on natural antibody diversity and includes both single and multiple (up to 14) CDR mutations. We demonstrate how this library design approach can be used along with yeast surface display to identify stable and specific variable domains with increased affinity.To evaluate potential solutions to these challenges, we have sought to identify mutations that increase the affinity of a camelid single-domain antibody specific for the C-terminus of α-synuclein  Figure . This van Figure , it is rToward our goal of developing systematic and robust affinity maturation methods, we first sought to identify permissive sites in the CDRs of N2 that weakly impact antibody affinity using both computational and experimental methods. Two of the CDRs (CDR2 and CDR3) are involved in mediating antigen binding Figure . Our comHH concentrations  and eight in CDR3 . The other seven mutants that displayed greater reductions in binding included five CDR2 mutants  and two CDR3 mutants (Y100 and W100e), which were not subjected to further mutagenesis. Four of the disruptive mutations (N52 and K56 in CDR2 and Y100 and W100e in CDR3) were identified in our computational alanine scanning mutagenesis . These and other previous results . SDS-PAGE analysis revealed high purities . The relative binding of each mutant was evaluated using fluorescence polarization at three V results , 53, 54 7 variants to enable 10-fold oversampling of the library using yeast surface display given that our typical yeast transformation efficiencies are ~108 transformants. Second, we aimed to generate a single library with all possible combinations of wild-type residues as well as single and multiple mutations at the 11 permissive sites in CDR2 and CDR3 as well as at three additional sites not tested during alanine mutagenesis . This limits the number of possible mutations at each CDR site to typically one to two mutations in addition to the wild-type residue. Third, we sought to sample mutations that most closely correspond to those observed in the CDRs of natural antibodies in a site-specific manner. To accomplish this, we used the AbYsis database to identify the most common amino acids in camelid VHH and human VH domains at each site in CDR2 and CDR3  members of the initial library, and the results are summarized in Figure Based on these four key objectives, we designed the library shown in Figure S. cerevisiae and screened for variants with increased affinity for the α-synuclein peptide. The sorting process involved five rounds of selection via magnetic-activated cell sorting (MACS) with progressively reduced concentrations of α-synuclein peptide (starting at 50 nM peptide and ending at 5 nM) and one additional round of selection via fluorescence-activated cell sorting (FACS) (20 nM peptide). The sorting process was continued until the antigen binding of the library was increased by at least fivefold relative to wild type, as judged by flow cytometry. Selections were performed in a buffer (PBS) that contained both BSA (1 mg/mL) and milk (1% w/v). We have found previously that antibody selections in complex environments  lead to identification of antibodies with improved specificity  with 1–6 mutations in CDR2 and CDR3. Sequence logos in Figure The enriched VHH variants that were identified in the enriched library. The variable domains expressed at levels (purification yields of 0.1–2.0 mg/L) that were generally similar to wild type (1.0 mg/L), and also displayed purities similar to wild type . We first used fluorescence polarization to evaluate the affinities of the variable domains for the α-synuclein peptide  was approximately threefold lower than the previously reported value (DK of 190 ± 30 nM) that was measured by isothermal calorimetry . Both variable domains displayed improved affinity , while the N2.12 variant displays similar non-specific binding as wild type . These results demonstrate that the affinity-matured VHH domains display similar or improved specificity relative to wild type.We also evaluated the specificity of the N2.12 and N2.17 VHH variants that were isolated during the sorting process  display similar stabilities as wild type (apparent melting temperature within 1°C of wild type). The variable domains with the largest reductions in apparent melting temperature  had the highest number of mutations (5–6 mutations). A direct comparison of affinity versus stability for the VHH domains reveals a wide range of affinity/stability trade-offs  to evaluate the impact of the acquired mutations on affinity, stability, and specificity. Six single reversion mutants were created for N2.12, while four single reversion mutations were created for N2.17. The purities of the reversion mutants were similar to wild type .To better understand the origins of the strong and weak trade-offs between affinity and both stability and specificity for the selected VAK). Reversion mutations that reduced affinity and/or stability—which signifies that the original mutations increased affinity and/or stability—correspond to reduced AK or apparent melting temperature p-value of 2 × 10−5; Figure p-value of 0.67; Figure HH domain that we obtained, as the large affinity enhancement (>7-fold) is achieved without compromising stability . This reversion mutational analysis also reveals that the affinity enhancement of N2.12 is largely due to four mutations . The S96 mutation is particularly interesting because it contributes positively both to affinity and stability, as judged by the fact that the reversion mutation (F96) reduces both properties . By contrast, the R53 mutation increases affinity  at the cost of stability , and the W52b and T100d mutations increase affinity  without significantly impacting stability .The affinity and stability measurements are summarized in Figure tation G4 is highltation G4 is highlHH domain (N2.17) revealed key differences relative to the less stable N2.12 variant . The three key affinity mutations —which were also observed in the less stable N2.12 domain—had little impact on stability (<1°C). These findings highlight that the affinity/stability trade-offs observed in our enriched library can be addressed either by screening a sufficient number of VHH variants or by performing reversion mutational analysis to identify destabilizing mutations that are not required for affinity.Reversion mutational analysis of the more stable Vt Figure B. None op-values <0.01; Figure p-value of 9.4 × 10−6; Figure p-values <1.1 × 10−5; Figure The specificity of the reversion mutants was analyzed by evaluating their relative propensity to interact with milk proteins Figure . A decreHH affinity, we performed computational modeling of two of the mutant variable domains (N2.12 and N2.17). This was accomplished by introducing the corresponding mutations into the crystal structure of the wild-type N2 domain in complex with the α-synuclein peptide (PDB: 2X6M) and relaxing the structures via CHARMM force field energy minimization (via an interaction between the backbone amide in the antibody (I55) and carboxylate oxygen in the antigen (E137). However, this does not appear to explain the increased affinity of N2.12 because the mutation increases the interaction distance (2.6 Å) relative to wild type (1.7 Å). Instead, the increase in affinity for N2.12 appears to be due to indirect effects that involve enhancement of existing interactions as well as introduction of new interactions that involve wild-type CDR residues  are necessary to achieve large >5-fold) gains in affinity for the N2 Vfindings , 3, 5, t-fold gaifindings , 64–66. findings , 67, 68.findings , 67, 69.There are multiple considerations related to our natural diversity mutagenesis approach that deserve further consideration. First, the primary problem during affinity maturation is obtaining mutations that increase affinity but reduce specificity. Our use of natural antibody diversity to guide library design—which has been reported previously in related ways by others –41, 70—aIt is also notable that our mutational approach was useful for identifying beneficial mutations in the highly variable CDR3 in addition to the less variable CDR2. Two of the key affinity mutations in both N2.12 and N2.17—F96S and S100dT—were in CDR3. The most common residues at many sites in CDR3 occur at relatively low frequency (13–21% for positions 95–100 g). Therefore, it was not obvious that sampling such a small number of natural diversity mutations (1–3 mutations per site for nine sites in CDR3) in such a highly diverse CDR would be sufficient to identify affinity-enhancing mutations. For example, the natural occurrence of the wild-type residue (Phe) at position 96 in CDR3 is 4% (combined human and camelid diversity), and we sampled only one mutation (Ser) at this site that is also relatively uncommon (9%) despite being more common than most other residues at this CDR3 site. Likewise, we sampled three mutations at position 100d in CDR3  that were all relatively uncommon (5–11%). Nevertheless, we identified a beneficial mutation (Thr) that occurs relatively infrequently (5%) at this site in CDR3. These results suggest that natural diversity mutations in CDR3—especially for affinity maturation—may be particularly useful for libraries aimed at isolating combinations of mutations that result in large increases in affinity without over enrichment in highly interactive residues that are likely to also mediate non-specific interactions.Despite the strengths of our natural diversity mutagenesis approach, one obvious weakness is related to the use of inexpensive primer synthesis methods that rely on standard degenerate codons to generate libraries. This results in the limitation that some combinations of wild-type CDR residues and the most common natural diversity mutations are (i) not possible, (ii) require too many additional mutations to justify including them, and/or (iii) require inclusion of undesirable codons . While we allowed a cysteine mutation at one position (100c) to maximize natural diversity coverage, it is undesirable to include too many cysteine mutations due to complications associated with unpaired cysteines.An example of the limitations of using degenerate codons to generate antibody libraries is related to position 52b in CDR2. The wild-type residue at position 52b is Leu, and the two most common residues at this position are Lys  and Arg (22%). However, this requires sampling a minimum of six codons, which corresponds to a minimum of five residues and overrepresentation of arginine (two codons) to achieve natural diversity coverage of 56% (an average of ~9% per codon). Therefore, we sampled Gly, Val, and Trp in addition to the wild-type residue (Leu) at position 52b using four codons to achieve natural diversity coverage of 36% and similar average diversity per codon (9%). Likewise, the wild-type residue at position 96 in CDR3 is Phe. In order to sample Phe and the most common residue (Gly), this requires sampling a minimum of four codons that include Val (5%) and Cys (1.5%). Sampling these four residues would result in natural diversity coverage of 23% (an average of ~6% per codon). Instead, we sampled Ser in addition to Phe using two codons to achieve natural diversity coverage of 13% (an average of ~6% per codon). This approach allowed us to sample a similar amount of natural diversity per codon and eliminated the use of an undesirable codon (Cys). These examples highlight the limitations of using standard degenerate codons to achieve the highest possible coverage of natural diversity mutations. This limitation could be readily solved using more expensive trinucleotide synthesis methods.Our results also demonstrate that affinity/stability trade-offs are common during antibody affinity maturation. We and others have previously found that CDR mutations that increase antibody affinity can be destabilizing , 49, 51.ameworks  to co-seAnother notable aspect of our findings relates to the impact of affinity-enhancing mutations on antibody specificity. Specificity is arguably the most difficult antibody property to maintain or enhance during affinity maturation –44. ThisHH domain while maintaining or enhancing stability and specificity. These encouraging results will need to be evaluated for other types of single- and multidomain antibodies to evaluate their generality. It will also be important to develop computational methods to improve library design by optimizing natural diversity coverage while minimizing the number of mutations. This is relatively straightforward to perform at any given CDR site but it is more challenging to globally optimize with increasing numbers of CDR sites. Nevertheless, efforts in optimizing antibody library design are key to avoid oversampling abnormal CDR sequences that are unlikely to lead to high antibody stability and specificity in addition to high affinity. We expect that methods such as the ones we have demonstrated in this work will be useful for rapidly and systematically optimizing antibodies for a wide range of diagnostic and therapeutic applications.Our systematic approach for using natural antibody diversity to design libraries with combinations of single and multiple mutations with limited diversity at each CDR site is effective for increasing the affinity of a camelid VHH domain  that contained an N-terminal pelB sequence for periplasmic secretion. Single point mutations of N2 were generated via site-directed mutagenesis using PfuUltra II .The wild-type N2 gene was created using PCR-based gene synthesis . The amin Figure  was obtaHH domain to framework 2, CDR2 to framework 3, and CDR3 to the C-terminus of VHH domain. The DNA fragments overlapped each other by ~20 bases, which enabled the three DNA fragments to be combined in a final amplification step using terminal primers. The terminal primers contained flanking NheI and SalI restriction sites as well as 45 bases of homology on each end with the yeast display plasmid (pCTCON2).The N2 natural diversity library was created using overlap extension PCR to introduce mutations in portions of CDR2 and CDR3 . Mutagenesis was performed using degenerate codons at 14 sites in CDR2 and CDR3  via homologous recombination. This process was performed as described previously (600 of 1.2) for a single library transformation, more DNA (nine preparations of 4 µg PCR product and 1 µg digested vector), and electroporation at higher voltage . After the yeast cells were allowed to recover, the yeast library was grown in SDCAA  for 48 h, and aliquotted for storage at −80°C. The library transformation resulted in 2 × 108 transformants. To assess the quality of the library, a small amount of the yeast library culture (1 mL) was miniprepped  and transformed into electroporation-competent bacterial cells . Several (22) plasmids from the initial library were isolated and sequenced, and all were found to be unique.The N2 natural diversity library genes were ligated into the yeast display plasmid and transformed into eviously  with minHH fusion proteins, the medium was switched to SGCAA  and grown for 16 h at 30°C with agitation. The yeast medium was supplemented with ampicillin , kanamycin , and penicillin-streptomycin .The yeast cultures were first grown at 30°C with agitation in SDCAA to an OD value of 1–2. To induce the expression of Aga2-Vvia five rounds of MACS and one round of FACS. For each sort, yeast were washed twice with PBS containing BSA  and resuspended in a solution containing the biotinylated α-synuclein peptide (biotin-GYQDYEPEA) and PBS-B supplemented with 1% milk . For the FACS sort, 1,000× diluted anti-c-myc chicken IgY antibody  was added to this mixture to detect VHH display. The yeast and α-synuclein peptide solution was mixed end-over-end at room temperature for 2–3 h. Next, the cells were washed once with PBS-B and sorted for antigen binding.The natural diversity library was sorted via flow cytometry . The enriched yeast cultures after sorts 5 and 6 were miniprepped and subcloned into a bacterial expression vector (pET-17b). Several (~10) plasmids from each sort were isolated and sequenced.For MACS sorts, yeast cells were resuspended in PBS-B (5 mL) and mixed with Streptavidin MicroBeads . After incubation on ice (10 min), the yeast cells were pelleted and resuspended in PBS-B and passed through a MACS separation column . The column was connected to a MidiMACS separator magnet  that was attached to a MACS MultiStand . Next, the bound yeast cells were eluted by removing the column from the magnetic stand and flowing SDCAA (7 mL) through the column. The collected cells were then grown overnight in SDCAA (30°C) with agitation and subjected to additional rounds of sorting. For the FACS sort, yeast cells were resuspended in PBS-B (200 µL) with 100-fold diluted secondary reagents , and allowed to incubate on ice (5 min). The cells were washed once, analyzed, and sorted HH domains were expressed in bacteria  using auto-induction media (200 mL) supplemented with ampicillin (100 µg/mL) and chloramphenicol (35 µg/mL) (g (5 min) and filtered . Next, the VHH domains were refolded via buffer exchange  into 6 M GuHCl (pH 7.4). The antibody domains were allowed to equilibrate overnight (4°C) before being buffer exchanged into PBS (pH 7.4). Finally, the VHH domains were concentrated  and filtered again (0.22 µm filters). The concentrations of the VHH domains were measured via UV absorbance measurements at 280 nm. The extinction coefficients of the VHH domains were 27,180–32,680 M−1cm−1, which were calculated based on their amino acid sequences. The purity of the VHH domains was evaluated using SDS-PAGE analysis , and the gels were stained using Coomassie dye .V5 µg/mL) . After 4HH and variants thereof were measured using fluorescence polarization. The VHH domains were prepared at a range of concentrations (0.8 nM–1.6 µM) and mixed (75 µL) with the α-synuclein peptide labeled with a tetramethylrhodamine (TAMRA) fluorophore . The antibody–antigen mixtures were prepared in 96 well flat bottom black polystyrene plates . The binding buffer was PBS supplemented with BSA [0.001% (w/v)] and Tween 20 [0.001% (v/v)]. Background wells were prepared that contained the same concentration of TAMRA-labeled α-synuclein peptide without antibody. The antibody–antigen mixtures were allowed to equilibrate at room temperature for 3 h. Fluorescence polarization was then measured  at an excitation wavelength of 530 nm (5 nm bandwidth) and an emission wavelength of 582 nm (10 nm bandwidth).The affinities of the N2 VDK value using a four-parameter model that accounts for the fact that the antibody is not in excess of antigen at some of the evaluated antibody concentrations:min is the minimum fluorescence polarization value, FPmax is the maximum fluorescence polarization value, [Ab] is the total VHH concentration, [Ag] is the total antigen concentration, and DK is the equilibrium dissociation constant. The equation was fit using the Microsoft Excel solver tool to minimize differences—namely the sum of squared differences—between the data and the model. At least three independent experiments were performed for each VHH antibody.The fluorescence polarization raw signals were background subtracted and two replicates were averaged for each antibody concentration. The average data were then fit to determine the HH domains were determined using measurements of extrinsic fluorescence  as a function of temperature. Protein Thermal Shift buffer (5 µL), VHH domains (12.5 µL of 0.08 µg/µL VHH), and Protein Thermal Shift dye (2.5 µL of 8× solution) were mixed in opaque 96-well PCR plates and sealed with foil . The background samples were prepared with water (12.5 µL) instead of VHH domains. Thermal melts were performed using a LightCycler 480 real-time PCR instrument (Roche). The fluorescence  was measured as the plate was heated from 37 to 95°C. Many (>60) acquisitions were collected per 1°C, and the heating rate was ~0.6°C/min.The apparent stabilities of the VHH domains were determined by analyzing the first derivative of the fluorescence with respect to temperature. This involved fitting a second-order polynomial to the major peak and solving for the temperature at which the maximum occurred (or the minimum if the negative derivative is used). The reported melt curves were background subtracted using background signals obtained without antibody. Next, the fluorescence data were subtracted by the relatively low signal at 50°C and divided by the maximum fluorescence signal . Finally, the pre- and post-transition regions of the normalized fluorescence data were flattened using linear fits  were coated with milk  for 8 h and then washed with PBS. The VHH domains were diluted to 1,000 nM in PBST, added to the well plates and allowed to incubate overnight at room temperature. The well plates were then washed with PBS and secondary reagents were added to detect bound antibodies. The second method evaluated the propensity of the purified antibodies to bind to six immobilized non-antigens . Non-antigen proteins were diluted in PBS  and immobilized in separate wells at 37°C for 1 h in 384 well plates. The wells were subsequently washed with PBST. Variable domains  in PBS with 1 g/L BSA and 0.1% (v/v) Tween 20 were added to the well plates and allowed to incubate at room temperature for 2 h.The specificities of the VHH was performed similarly for both specificity tests. Secondary antibody  in PBST was added, allowed to incubate for 1 h, and then washed with PBS. Next, the well plates were incubated with diluted horseradish peroxidase-conjugated goat anti-mouse IgG  in PBST for 1 h and then were washed with PBS. The bound antibody was detected by adding substrate , quenching after 20–40 min (25 µL of 2 M H2SO4) and measuring the absorbance values at 450 nm (Tecan Safire2 plate reader). Normalized binding signals were calculated as signal divided by background, and the background values were absorbance measurements without primary (VHH) antibody.Detection of bound VHH-antigen crystal structure (PDB: 2X6M) was energy minimized using the CHARMM force field and the adopted basis Newton–Raphson routine  in complex with antigen were also generated. These structures were simulated alongside the energy-minimized wild-type complex. We created the N2.12 and N2.17 variants using the Mutator program of IPRO suite of programs (HH variants. Only contacts within 5 Å were analyzed.Structural models of two affinity-matured Vprograms . This apprograms . This waKT, PT, RC, TL, and CM designed the research; KT and SS performed experiments; RC and TL performed computational analysis; SL performed bioinformatics analysis; and KT, RC, CM, and PT wrote the paper.PT has received consulting fees and/or honorariums for presentations of this and/or related research findings at MedImmune, Eli Lilly, Bristol-Myers Squibb, Janssen, Merck, Genentech, Amgen, Pfizer, Adimab, Abbvie, Abbott, DuPont, Schrödinger, and Novo Nordisk. All other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Covalently cross-linked gels are utilized in a broad range of biomedical applications though their synthesis often compromises easy implementation. Cross-linking reactions commonly utilize catalysts or conditions that can damage biologics and sensitive compounds, producing materials that require extensive post processing to achieve acceptable biocompatibility. As an alternative, we report a batch synthesis platform to produce covalently cross-linked materials appropriate for direct biomedical application enabled by green chemistry and commonly available food grade ingredients. Using caffeine, a mild base, to catalyze anhydrous carboxylate ring-opening of diglycidyl-ether functionalized monomers with citric acid as a tri-functional crosslinking agent we introduce a novel poly(ester-ether) gel synthesis platform. We demonstrate that biocompatible Caffeine Catalyzed Gels (CCGs) exhibit dynamic physical, chemical, and mechanical properties, which can be tailored in shape, surface texture, solvent response, cargo release, shear and tensile strength, among other potential attributes. The demonstrated versatility, low cost and facile synthesis of these CCGs renders them appropriate for a broad range of customized engineering applications including drug delivery constructs, tissue engineering scaffolds, and medical devices. Successful translation from bench concepts to clinic products is often limited by the few materials available for device and formulation development that can be easily processed and quickly pass regulatory hurdles. Furthermore, few formulations and materials platforms are dynamic and diverse enough to fulfill structural, manufacturing and therapeutic needs of a product. Covalently crosslinked thermosets provide unique advantages for structural engineering by offering robust mechanical properties but often rely on complicated processing conditions using harsh solvents, high temperatures, or toxic catalysts that preclude direct implementation in biomedical applications . Alternaffolding . Howeverffolding , especia systems . While tMotivated by the emergence of vitrimers and concepts of dynamic thermosets  and covain vitro or in vivo aqueous environments. This process is designed to be accessible to all minimally equipped biomedical engineering labs and represents a new class of materials able to provide broad functionality in a diverse set of applications ranging from drug delivery to food science. Innovations in drug delivery and device engineering will benefit from the reproducible and robust manufacturing methods that employ readily available feedstocks to construct functional novel materials.This report explores three variations of the caffeine-catalyzed gel (CCGs) concept , with a 22.1Reagents and compounds from the FDA's Generally Regarded as Safe (GRAS) materials list were selected as candidates for forming bioresponsive bonds such as esters, amides, ethers, or thioethers. Additionally, well-defined, inexpensive oligomers of common biomedical engineering polymers were selected to provide an easily translatable platform applicable to a broad set of polymer feedstocks. Focusing on simple acids derived from plant and food sources such as citric, succinic, and malic acids, we used caffeine, as a mild base to deprotonate the carboxylic acids. The resulting carboxylates can ring-open epoxides to form ester bonds using solvent-free conditions.All reagents were purchased from Sigma Aldrich unless otherwise noted and used as received. Citric acid and caffeine were stored in a desiccated environment to control moisture. Poly(ethylene glycol) diglycidyl ether , poly(propylene oxide) diglycidyl ether  were stored in sealed opaque containers in 4 °C refrigerators.2PO4 and 0.896 g of NaOH were dissolved into 1 L of deionized water and titrated to reach a pH of 6.8. Phosphate buffered saline was prepared by dissolving PBS preparatory packet from Aldrich in 1 L of deionized water and stored as a stock solution until use.Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared as 1 L stock solutions according to US Pharmacopeia recommendations, stored at room temperature (approximately 21 °C) until use. To prepare SGF, 2 g of dry NaCl was dissolved into 1 L of deionized water. The solution was titrated using concentrated HCl to reach a pH of 1.2. To prepare SIF, 6.8 g of KH2.2All reagents were directly measured into a flask equipped with a stir bar, heated to 70 °C, and well mixed to form a homogeneous viscous pre-polymer. Shortly after reaching the gel point, the viscous reaction mixture was poured into silicone molds and incubated at 90 °C to accelerate extensive cross-linking. Temperatures and times for pre-polymer formation and final gel cross-linking density were customized based on reagents and application needs. The rate of network formation can be further accelerated with higher catalyst concentration or higher heat, or by using anhydrous conditions. The ability of selected reaction conditions to form complete crosslinked networks was validated by performing an extraction on representative CCGs and evaluating residuals, ensuring only the catalyst is removed in this experiment.For the PEG.CA gels specifically, dry caffeine  and citric acid  were measured into a 20 mL vial equipped with a Teflon lined stir bar. Diglycidyl ether poly(ethylene glycol)  was added directly to the powders and the entire mixture was incubated at 70 °C and stirred at 350 rpm for 5 min until well mixed. The reaction was allowed to proceed to its gel point as observed in a rapid increase in viscosity. At this time the mixture was poured into a mold of choice and transferred to a convection oven at 90 °C. After 24 h the pieces were removed from the oven for analysis. At this point no post-processing was required as it was found all reagents were reacted into the network and no residual material was left behind. All other materials were synthesized using the same protocol as described above, maintaining molar ratios and adjusted for mass amounts.2.3Infrared spectra were recorded on an ALPHA FT-IR spectrometer (Bruker) and analyzed using OPUS v. 6.5 software (Bruker). Spectra were recorded at several time points during gel formation. Each time point represents an aliquot taken from a single continual polymerization and was directly measured on the ZnSe ATR crystal as either a liquid, gel, suspension or solid depending on the time at which the sample was measured. For example, at 15 min the polymerization was not crosslinked and still a liquid, therefore the liquid was directly dispensed on the crystal but at 600 min the mixture was a solid, thus a thin slab of polymer was directly placed on the crystal. Background spectra were recorded between samples and the crystal was cleaned with isopropanol.2.41H NMR spectroscopy analysis was performed on an AVANCE 400 spectrometer (Bruker) with a 5 mm PABBO BB/19F-1H/DZ-GRD z10861810556 probe. Spectra were analyzed using MNOVA software (Mestrelab Research) and referenced in ppm to the solvent DMSO‑d6. Samples were directly removed from the polymerization and placed in an NMR tube and dissolved or swelled in DMSO‑d6 before NMR analysis.2.5Surface morphology of textured materials was visualized by the means of a JEOL 5600LV (Jeol Inc.) scanning electron microscope (SEM). For visualization under SEM, samples were fixed to aluminum stubs with double-sided adhesive carbon conductive tape and subsequently sputter-coated with carbon using a Hummer 6.2 Sputter System (Anatech USA).2.6Fabricated surfaces were characterized for the degree of hydrophobicity by taking the static contact angle measurements by the means of the Krüss Drop Shape Analyzer DSA100 with the software Drop Shape Analyzer . Contact angles of water droplets over the sample surface, fixed to lay flat on a horizontal plane, were measured at room temperature. A fixed volume of 250 μL was dispensed onto the substrate, and then the contact angle made between the line tangent to the liquid droplet and the substrate surface was measured. The macroscopic droplet profile was photographed by a camera installed within the instrument. Each surface was analyzed in 8 different regions distributed across the plane of the surface. The average and the standard deviation values for each surface were calculated and reported.2.7Small discs (2 mm H ⋅ 10 mm W) of PEG.CA, PEG-PPO.CA, and PPO.CA materials were incubated in simulated biological fluids (simulated gastric fluid (SGF), simulated intestinal fluid (SIF), phosphate buffered saline (PBS)) and organic solvents  at room temperature (approximately 21 °C). Hydration kinetics and solvent response averages were calculated based on mass change per unit volume of representative gel formulations and represent an n = 3. For each solvent evaluated, the initial mass of a gel piece was recorded and taken as t = 0. The experiment commenced upon addition of the solvent of choice directly to the gel under study in a 15 mL conical tube. All pieces were incubated at 37 °C, rotated at 150 rpm while tilted at an angle to allow maximum mobility. At each time point the piece was removed from solution, blotted dry to remove surface water, and measured for mass immediately.2.8D412: 33 mm gauge length x 3 mm width x 1.5 mm thick were manufactured. Quasi-static test-to-failure (0.05 mm/s displacement rate) was conducted for n = 10 samples within each group on an Instron 5900 mechanical testing system using a 50 N load cell. The elastic modulus of samples was calculated by fitting a best-fit linear line to the stress-strain curve prior to rupture. For compressive stress-strain testing, standard cylindrical shapes molded after ASTM D695: 12.7 mm diameter x 25.4 mm height were manufactured and fitted on an Instron machine. The compressive modulus was calculated by differentiating the stress-strain curve. For shear testing standard disc shapes molded after ASTM D6204: 25 mm diameter x 2 mm thick, were manufactured. Shear stress – shear rate curves (shear strain = 5%) were generated for n = 10 samples per polymer on a TA rheometer. The shear modulus was calculated by differentiating the stress-strain curve.Tensile, compressive, and shear properties of the polymers were characterized using standard techniques as defined by the American Society for Testing and Materials (ASTM). Testing shapes were cured by injecting viscous fluid into 3D printed molds. In all cases width, thickness, and gauge length of each sample was measured prior to testing using digital calipers. For tensile testing standard dumbbell shapes modeled after ASTM 2.9To demonstrate self-healing, CCGs were ruptured at the midpoint of a dumbbell-shaped specimen as described above, through controlled tensile failure. Following rupture, the samples were placed back into PDMS molds and incubated at 90 °C in an oven for 12 h. Following self-healing the samples were stored at room temperature for 48 h prior to performing mechanical characterization. The tensile properties of specimens was determined using the quasi-static tensile test protocol described above.2.10® software .Thermal properties of CCGs were evaluated using a DSC 8000  for 3 cooling/heating cycles from −50 °C to 180 °C at a rate of 10 °C/min with a 2 min isotherm at 180 °C. Samples were sealed in analytical aluminum pans and balanced against a reference pan with no contents. All samples evaluated were 8 mg or higher. Data was analyzed using Pyris2.113, 16 × 103, 16 × 103 and 2 × 104 cells/well respectively. Cells were obtained directly from ATCC and were tested negative for mycoplasma. HeLa and HEK293 cells were cultured in 100 μL DMEM containing 1% non-essential amino acids, 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (Life Technologies) per well. C2BBe1 and HT29-MTX-E12 cells were cultured in the same medium but was additionally supplemented with 4 mg/mL human transferrin (Life Technologies). Cells were kept in culture for 3 days before replacing the medium, to which the dissolved aqueous polymer solutions were added . After 72 h, cytotoxicity was quantified by adding 10 μL alamarBlue reagent (Life Technologies) to each well. Absorbance at 570 nm was recorded on an Infinite M200Pro (Tecan) using 600 nm as reference wavelength. Following common practice, n = 8 biological replicates were recorded for each group. A positive control was provided by lysing cells with 1% Tween-20 and cells that were not subject to any polymer provided a negative control.Polymers were completely degraded in dilute HCl pH 2. Upon confirmation of complete dissolution, the solutions were adjusted to pH 7.0 using NaOH. The final polymer solution was diluted with Dulbecco's Modified Eagle Medium (DMEM) (Life Technologies) to 50 mg/mL before testing. Cytotoxicity was tested on HeLa, HEK293, C2BBe1 (ATCC) and HT29-MTX-E12 cells  by seeding them in a 96-well plate at a density of 6 × 102.12in vivo toxicity of the polymers, we followed the guidelines for toxicity tests as outlined by the FDA [2 asphyxiation and necropsied. Thymus, heart, lungs, liver, spleen, adrenal glands, kidney, stomach, small intestine, large intestine, ovaries and uterus were extracted weighed and sectioned for histopathological examination. An independent pathologist examined all organs to detect any abnormalities in a blinded fashion. All animal procedures were conducted in accordance with protocols approved by the Massachusetts Institute of Technology (MIT) Committee on Animal Care.In order to test  the FDA ,30. Spec2.13Drug was added to the polymerization mixture prior to addition of the diglycidyl ether monomer. To model drug release from CCGs, analyte containing polymers were molded to 10 mm discs. n = 8 replicates were recorded for each condition. These were placed into a single fluid volume of 10 mL in simulated gastric fluid (SGF) or simulated intestinal fluid (SIF), incubated at 37 °C, and rotated at 150 rpm. Samples were immersed in 10 mL fluid each to simulate an infinite sink for elution. At each time point, 100 μL samples were removed from the stock solution and stored frozen until analysis. Release experiments were recorded as cumulative release and all concentrations were standardized against blank gel formulations to account for background interferences in the chosen detection methods. In addition, analyte stability was evaluated for each compound as a free solution of the theoretical ½ max concentration. For each 200 mg piece at a 40 wt% loading it was assumed 80 mg of compound could be potentially released. Drug concentrations were analyzed directly from the aliquoted solutions on an Infinite M200Pro (Tecan) multi well plate reader using absorbance at 345 nm for piperaquine or using excitation and emission wavelengths of 490 nm and 525 nm for FITC-dextran.33D cross-linked networks formed readily by using a mixture of multifunctional acids and epoxides, such as the commercially available diglycidyl ether oligomers of poly(ethylene glycol) (PEG.CA) a and pol2O for 24 h and analyzing the solution using 1H NMR spectroscopy to detect any soluble compounds. The 3D devices are capable of retaining their engineered shapes at room and body temperature as determined during handling in ambient conditions by actions such as twisting, squishing and stretching b. An excCCG based materials can be reshaped by catalyzing bond exchange via applied pressure and temperature. To gauge the rate of bond exchange occurring at 90 °C, three rectangle strips of polymer from a single batch polymerization of each CCG chemistry were individually pinned in the same horseshoe configuration and incubated at 90 °C for different lengths of time . Pieces 1H NMR spectroscopies (−1 increased in intensity with time signifying the formation of polyester bonds. Simultaneously a peak at 12.5 ppm in the 1H NMR spectra decreased in intensity over time supporting the formation of ester bonds by consumption of the carboxylic acid protons in the acid monomers.The formation of the poly(ester-ether) networks was observed using FT-IR e and 1H oscopies f. Here t2O) on the native flat surfaces of CCG samples showed little difference with regard to the oligomers used , simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) extensively d, but alTensile, compressive, and shear properties of the polymers trended in accordance to the internal chemistry of the networks a–d. An ig of the CCG materials (in vitro cell toxicity showing low cytotoxicity in four cell lines (50) was similar across all cell lines and CCG variants, ranging between 2.96 and 5.90 mg/mL. The HT29-MTX-E12 cell lines showed an even higher LC50 however with greater variability, ranging from 6.04 to 13.84 mg/mL, likely due to the protective mucus layer being produced by the cell lines. Furthermore, bulk in vivo administration of the CCGs to three rats per CCG chemistry showed no symptoms of toxicity, supporting direct use of CCGs in oral administration. In all cases test animals showed no significant weight changes, no behavioral abnormalities, and no gross or histopathological organ changes  and alkaline (SIF) pHs each . MacromoThe good solubility of hydrophilic piperaquine yielded a burst release for both materials c. Howeve41H NMR spectroscopy experiments showed the gradual development of poly(ester-ether) networks by simultaneous formation of ester bonds and consumption of carboxylic acid protons from citric acid. This mechanism is compatible with numerous combinations of multi-functional glycidyl ether and acid containing monomers as well as inclusion of anhydrides or lactones, opening the opportunity to construct an incredibly diverse array of customized materials based on specific application needs. The simple manufacturing method with no residual solvents or toxic catalyst allow for these materials to be cast in molds of many shapes with the capacity to retain micron sized features with no measurable shrinkage or distortion and ready for immediate application, advantageous for accelerated translation and regulatory evaluation.Herein we introduce a simple readily accessible synthesis and engineering platform for the formation of 3D cross-linked polymer gel using caffeine as a catalyst. Time dependent FT-IR and CCGs are stable at room temperature with the inherent capacity to be reshaped or healed representing a new class of biocompatible, dynamic thermosets that can be engineered for advanced customized applications. The shaping and healing capability is imparted by the inclusion of excess carboxylic acids in the formulation, which allow ester exchange to be easily catalyzed under heat and does not require addition of new materials or introduction of complicated interfaces, maintaining the simplicity of this platform. Shape changing or healing is time dependent and scales with the number of bonds required for rearrangement to completely relax applied stress or bridge interfaces.in vitro as well as no detectable in vivo toxicity after large dose oral administration in rodents we believe this platform will be useful in high-throughput evaluation of cast moldable biomedical devices intended for therapeutic applications.Finally, hydration kinetics and mechanical testing demonstrate how the chemistry of CCGs can be tuned by changing the oligomeric backbone building blocks, empowering designer customization based on application specifications. Differences in solvent response enable the drug release profile of CCGs to be tailored with potential to further customize by including chemically diverse crosslinking monomers that feature groups capable of advanced environmental response such as ionic coordination, or disulfide bond formation. Impressively, the mild chemistry used for synthesis of CCGs permits direct loading of compatible active ingredients at the onset of device formation, providing a reproducible, efficient manufacturing method for high cargo loading. Given the highly tunable release properties, evidence of low cytotoxicity on multiple cell lines 5In summary, we've demonstrated a method using caffeine and simple polyester chemistry to synthesize biocompatible covalently cross-linked gels in a one-pot system. These CCGs represent a versatile synthesis platform utilizing simple modular chemistry that can be tailored in accordance to application needs to provide broad functionality in a diverse set of applications ranging from drug delivery to food science.


C-isotopic chemostratigraphic correlation combined with biostratigraphic constraints show that the Chaqiela section spans early Aptian through early Campanian period, and that almost all of the carbon cycle perturbations and Oceanic Anoxic Events during the mid-Cretaceous period are well recorded in the continental margin area of the southeastern Tethys Ocean. Significantly, two levels of methane-derived authigenic carbonates were identified at the onset of OAE1b near the Aptian-Albian boundary. We suggest that an increase in methane release from gas hydrates, potentially driven by sea-level fall and bottom water temperature increase, may have contributed to the large negative δ13Corg excursions and global warming during OAE1b.The organic carbon isotope (δ Theold snap 61113. A boundary 111213. H13Corg excursion in the middle part of the Gambacunkou Formation is identified as the latest Cenomanian OAE2 based on the regional and global C-isotope correlation and the foraminiferal stratigraphy in Gamba and Tingri area  boundary have been established at Gamba and Tingri .The large positive δgri area , Fig. S413Corg excursion, from −25.9‰ to −23.3‰, is located at the lower part of the Zongshan Formation 381413Corg excursion of SCBE in the Cheqiela section (2.6‰) is far larger than the δ13Ccarb records in the English Chalk sections (~0.5‰). The large amplitude shift of δ13C during SCBE is also recorded in skeletal calcite of mollusk in southwestern British Columbia, CanadaThe last large positive δormation . The limormation 192123242ormation 18, indicgri area  and soutgri area  suggest 13Corg excursions in the Chaqiela section. The one located at the lowermost part of the Gambacunkou Formation, from −25.7‰ to −24.7‰, may correspond to the OAE1d interval in the latest Albian  in the Chaqiela section . The MDAC-II (13Corg shift of 1.8‰ (from −24.9‰ to −26.7‰). The MDACs were also found underneath and within the OAE1b black shale level in southern France13C excursion in both δ13Ccarb and δ13Corg during OAE1b interval.The release of  section  and 2. Te MDAC-I  and 2B,  MDAC-II  and 2A, 13C anomaly during OAE1b was also documented in terrestrial wood fragments , compare to the terrestrial plant records (~1–1.5‰), implies that the majority of methane released in continental margin area of the southeastern Tethys Ocean was dissolved in the water column. Only part of methane entered into the atmosphere and influenced global carbon cycle and climate.The negative δragments 37, leaf 13C excursion of OAE1b during Aptian-Albian transition period40414 generation and the formation of gas hydrate.Gas hydrate dissociation can result from pressure decrease due to sea-level fall or temperature increase of bottom water3813Corg were analyzed on a ThermoFinnigan Flash Elemental Analyzer coupled in continuous flow to a ThermoFinnigan MAT 253 Mass Spectrometer. The δ13Corg values are expressed in per mil (‰) relative to VPDB standard.The rock samples were ground to powder with a mortar and pestle and then weighted and decarbonated with 10% HCl to remove all inorganic carbon. The samples were subsequently washed several times with deionized water to remove the HCl traces and then dried in an oven at 50 °C. Total organic carbon (TOC) and δHow to cite this article: Zhang, X. et al. Mid-Cretaceous carbon cycle perturbations and Oceanic Anoxic Events recorded in southern Tibet. Sci. Rep.6, 39643; doi: 10.1038/srep39643 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.