The CB[n]s with n = 5–7 have also been studied with 129Xe-NMR. They bind the noble gas with a large range of exchange rates. Starting with insights from conventional direct detection of bound Xe, this review summarizes recent achievements with chemical exchange saturation transfer (CEST) detection of efficiently exchanging Xe in various CB[n]-based supramolecular systems. Unprecedented sensitivity has been reached by combining the CEST method with hyperpolarized Xe, the production of which is also briefly described. Applications such as displacement assays for enzyme activity detection and rotaxanes as emerging types of Xe biosensors are likewise discussed in the context of biomedical applications and pinpoint future directions for translating this field to preclinical studies. Cucurbit[ The self-assembly of different molecules or likewise between molecules and single atoms may occur spontaneously and lead to the formation of different supramolecular structures with useful analytical features. These structures are held together by one or more types of interactions between the assembling components and the surrounding media, e.g., Hydrogen bonding, van der Waals interactions, hydrophobic and electrostatic interactions, donor-receptor effects, π-π stacking, or metal coordination, respectively . The gen129Xe is a valuable NMR probe to explore the formation and accessibility of such confined spaces for sensing the assembling status because it combines several useful features: Xe participates in weak, non-covalent interactions, thus it does not significantly disturb the interaction of the supramolecular constituents;129Xe is rather large and provides a sensitive measure for changes in the immediate molecular environment even without engaging in more stable  interactions;the NMR chemical shift range of the noble gas can be “hyperpolarized” (hp), i.e., its spin magnetization can be artificially enhanced for significantly improved NMR sensitivity;in combinations with its sufficient solubility in many solvents, including water, this enables straightforward NMR detection of this monoatomic reporter for biochemical targets and many other solution samples of interest.In any case, systematic understanding and monitoring of the assembly/disassembly process is an important aspect and requires methods to interrogate supramolecular systems. Optical detection is used in many studies because the fluorescence properties can significantly change between the assembled state and the free constituents. However, it can only be followed in sufficiently translucent samples and requires (switchable) fluorescent properties of one of the constituents itself or sufficient interaction with a dye. The latter one comes with a certain molecular size and thus requires a suitable interaction site within the supramolecular system. In many cases, confined spaces are involved in such systems - either in one of the constituents (as for molecular containers that bind another unit) or in the product . Particularly the small cavities are challenging when searching for a reporter with sufficient access but not too tight binding that might disturb the supramolecular assembly under investigation. Here, a monoatomic probe is ideal to interrogate such systems. This can be achieved through NMR with nuclei that are sensitive to their immediate molecular environment. Moreover, NMR is the method of choice for opaque conditions. To this end, 129Xe-NMR can deliver important insights into the properties of various supramolecular systems.Altogether, p-sulfonatocalix[n]arenes with lucigenin (as fluorescent guest) is an example with a high binding constant (~107 M−1) and strong fluorescence quenching. Such indicator displacement assays have been applied to analyze several types of analytes, including citrate, glucose-6-phosphate, inositol-1,4,5-triphosphate, tartrate, malate or nitrate α-amino acids, respectively urils (CB[n]s) s) . STAs ar(CB[n]s) . Introdu(CB[n]s)  or subst(CB[n]s) ,33.129Xe also represents an implementation of the appealing concept of a “label free” approach because the reporter is not covalently attached to one of the constituents. This review summarizes several NMR techniques for studying different supramolecular assemblies with a particular focus on cucurbit[n]urils as hosts in hp 129Xe-NMR spectroscopy. The principles of chemical exchange saturation transfer (CEST) with hp 129Xe as well as considerations regarding different system parameters and saturation-based detection schemes will be discussed as an example for high sensitivity Xe-NMR. Investigation of different members of the cucurbit[n]uril family will be elaborated to demonstrate the importance of the exchange kinetics. The translation potential of such host-guest based supramolecular assays to in vivo studies will be duly considered, followed by some suggestions to improve such 129Xe-based NMR/MRI investigations in future studies.Extending the displacement assay concept from fluorescence to NMR is an example for emerging concepts in supramolecular systems. As such, the use of T~300 K) and yields very small numbers (O(10−4)) due to the fact that the thermal energy linked to the Boltzmann constant 1H-NMR of biochemical samples suffers from the background interference arising from abundant nuclei when water has to be used as a solvent. Generally, millimolar concentrations of samples are required to achieve a decent NMR signal for various investigations. This holds in particular for acquiring multidimensional spectra or spatially resolved image (MRI) data. In certain cases, enrichment of different hetero nuclei, e.g., 15N, 13C, 31P, 19F, etc. has led to a surge in detection sensitivity of specifically labelled molecules in both solution and solid state NMR. Smaller supramolecular assemblies can be investigated in solution state while solid state NMR (ssNMR) is beneficial for studying larger supramolecular assemblies. The latter applies to biomaterials, proteins, metalloproteins s. Additionally, both C- or endo-shaped and S- or exo-shaped methylene-bridged glycoluril dimers are regarded as the important building blocks for producing different CB[n]s and their related derivatives [n]s are synthesized via the condensation reactions between glycoluril and either formaldehyde or paraformaldehyde in acidic  or refluxing conditions. CB[n]s typically comprise n = 5–10 glycoluril units that are linked via the two methylene bridges available on each side of the glycoluril. However, the synthesis yields a mixture of CB[n]s and CB[6] remains the predominantly isolated product, e.g., as a mix of CB[5] (10%), CB[6] (60%), CB[7] (20%) and CB[8] (10%)  (10%) ,79,80. n]s  [n] synthesis are 1) dimer formation, 2) creation of oligomer ribbons, and 3) the final ring closure [n]s mixtures by altering the reaction conditions, e.g., glycoluril and formaldehyde condensation in 9 M H2SO4 at ~75 °C for 24 h, followed by 12 h treatment at 100 °C [n]s mixture could be categorized into two parts [n” members such as CB[6], iCB[6]  such interactions are less pronounced (weak interactions) resulting in an enhanced water solubility [iCB[6] and iCB[7], are also isolated as kinetic intermediates while synthesizing CB[n]s in HCl (85 °C) [n]s achieved after synthesis is highly dependent upon the solvent and temperature applied during the condensation reaction  ), CB[8] lubility . Additio (85 °C) . The perreaction .n] family, e.g., CB[6], CB[8] and CB[10], are chemically inert and their poor solubility in common solvents hampers further applications. To some extent, the solubility of these members might be enhanced by utilizing cations or positively charged guest molecules [n]s with units that could enhance the solubility of CB[n]s in common solvents and also provides a handle that is suitable for further functionalization [n]s such as cyclohexano-substituted (CyH)nCB[n]s nCB[6] nCB[n]s) displayed higher solubility in both water and certain organic solvents compared to other substituted CB[n]s. For example, 1,4-DiCyHCB[6] indicated higher solubility in water while preserving similar host-guest properties compared to unsubstituted and almost insoluble CB[6]  fpentano ), Ph2CB phenyl ), HMeCB phenyl ), TMeCB phenyl ), (CyH)[2CB[6]  and (Me)nCB[6] , respecle CB[6] .n]s bind different guests including small organic molecules, amino acids, peptides and proteins [n]s attracts neutral hydrophobic molecules for encapsulation, while the two identical carbonyl rims entertain positively charged groups, e.g., ammonium groups or other cations. The binding process is also regulated by the size and shape of the guest molecules [n]s are anticipated upon reaching a guest volume of ca. 55%. For example, a highest binding affinity of 7.2 × 1017 M−1 was reported for CB[7] with a diamantane diammonium as guest. Such high guest binding affinity promotes CB[n]s applications in drug delivery s is generally investigated using different spectroscopic and analytical techniques. Regarding spectroscopic methods, NMR provides valuable information of the host-guest interaction (inclusion complexes) by means of changes in the observed NMR chemical shifts  was observed due to the large deshielding experienced by the H3 pointing toward ureido oxygen, while H1 and H2 appear at 3.3 ppm and 4.6 ppm, respectively, in the gas phase measurement. The observed chemical shifts of the CB[n]s protons are independent of the number of glycoluril units present in its isolated state s. The calculated NMR spectra indicated that CB[n]-based inclusion complexes upon solvation experience downshifts for the signals from H1 and H2 while the H3 shifts in opposite direction compared to free CB[n]s. Additionally, the orientation of the approaching guest to CB[n]s also shows a marked impact on the observed chemical shifts of the guest. For example, the ferrocene guest experiences a relatively large upfield shift of its protons upon complexation to CB when oriented parallely compared to inclusion complexes displaying perpendicular orientation of the encapsulated guests [The encapsulation of different guests by CB[ifts see a. For exed state . In the d guests . 1H-NMR is an essential tool for characterizing host-guest complexation formed by CB[n]s, a meager change in the chemical shift (ca. 0.5–1 ppm) often prevents its use in different applications. To overcome such issues, a reporter that can sense the interactions with guests with a well separated NMR chemical shift change (more than 30 ppm) at a significantly lower concentration (µM or lower) of the host will be of more interest. In this regard, hp 129Xe-NMR has emerged as a powerful tool for studying such supramolecular inclusion complexes with a pronounced chemical shift difference between bound and free Xe.Although conventional n]s, this limitation of conventional detection becomes especially problematic in combination with the low solubility of certain members of the CB[n] family. Long acquisition times become unavoidable even when an overpressure reservoir of pure Xe is available on top of a CB[n] solution in a sealed sample. The fraction of 129Xe can be enriched to ca. 80% , but this provides only a ~3-fold improvement in signal amplitude. NMR in general suffers from rather low sensitivity compared to other spectroscopic methods. While Xe as a mono-atomic gas with mM solubility in water  and othe2 into the sample directly after leaving the SEOP setup. One important advantage is that this comes with the option of practically unlimited signal averaging and very reproducible starting conditions in studies where an experimental parameter is incrementally changed over a whole series of acquisitions s for direct observation of Xe•CB[n] inclusion complexes with 129Xe-NMR. Conventional 1H-NMR would require significantly higher concentrations to reveal changes in the proton signals.The polarization values that are achieved with modern SEOP setups easily reach ous flow  and almoT1 relaxation will drive the spin system back to a tiny population difference that is impractical to detect. The favourable conditions thus exist only for a limited time window if no fresh Xe is re-delivered from the SEOP system. The delivery protocol and acquisition techniques have to be adapted to this condition. While continuous-flow operation allows in principle the stepwise acquisition of any complex data set, the repetitive delivery of Xe can become a limiting factor if delicate samples can handle the gas bubbling only to some extent. Typically, the gas is bubbled for 15–20 s at moderate flow rates (ca. 2 mL/min Xe flow rate) into a sample holder  and excessive bubbling should be avoided to keep foam formation in the sample under control. It should be noted that the hp state of the spin system is outside the thermal equilibrium and that lder see . The NMRn]s, however, have not shown any incompatibility with extended Xe bubbling. Solution volumes of ~1 mL should be bubbled sufficiently to achieve a Xe-saturated solution. Solvents with higher solubility for Xe  require longer gas delivery. Overall, the repetition time can add up to 30 s or more. Moreover, any magnetization that has been affected by the excitation pulse should be read out as quickly as it further decays. Spectroscopy applications that just screen for pools of bound Xe in CB[n]-derived hosts most efficiently use a 90° flip angle, followed by immediate Xe re-delivery.The bubbling delivery can become problematic when biological material such as proteins or cells are involved. Strong foam formation can occur as well as accelerated cell death . Synthetkout ≈ 10−1–10−2 h−1. This means that on average the host is still occupied for a long time after detection of the Xe guest. Re-delivered Xe would thus have lost its hp state already before entering the host for another detection because the life time of dissolved hp Xe from a He/N2/Xe mix is governed by T1 and amounts to ca. 120 s in water at 9.4 T  is an example  that is at 9.4 T . n]s with n = 5–7 are considered efficient hosts for small guests like 129Xe due to their matching cavity size. The commercial availability of them enables a quick evaluation of different CB[n] properties, e.g., molecular recognition and inclusion complexes using NMR without any hindrance. However, differential solubility of ‘odd’ 129Xe-NMR applications, a rather loose and reversible Xe binding is a prerequisite for achieving the desired Xe exchange rates, e.g., medium to slow exchange rates. This exchange of Xe is necessary for any measurements with hp 129Xe because the enhanced magnetization can be detected only once and needs to be replaced with freshly hp nuclei in cases where signal averaging is required . With CB[5] being the smallest host of this family, it accommodates 10CB[5]) with a moderate increase in solubility (ca. 0.7 mM at 293 K). Such solubility enhancement promoted the thermodynamics and kinetics-based investigations of Xe binding to CB[5]. Measurement of CB[5] using Xe-NMR revealed spontaneous incorporation of Xe into its cavity with a quite high binding constant and a low in/out exchange rate at 316 K. The 129Xe spectrum showed two peaks at 196 ppm (Xe in solution) and ~225 ppm (Xe•CB[5]), respectively. Interestingly, the encapsulated 129Xe showed a change in T1 from 15 ± 3 s to 28±5 s upon thermal cycling between 277 and 315 K. This relatively long T1 confirms the weak proton-xenon dipolar interaction existing between Xe and CB[5] in which the protons point outwards in addition to contributions from other relaxation mechanisms such as chemical shift modulation  was shown using MeCB[5] at 353 K in water compared to no binding of Xe in the solid phase. It was also found that CB[5] molecules interact with each other differently depending on the cavity being occupied or unoccupied with Xe, thus showing variations in the observed Xe chemical shifts. The Xe binding constant for CB[5] decreases with temperature. Detectability is better for per-hydroxylated CB[5] ((OH)dulation ,132. The129Xe, an NMR spectrum (single scan) of CB[5] (0.25 mM at 316 K) in the presence of less than 1 atm of Xe displays only one signal corresponding to free Xe in solution despite of having a high signal-to-noise ratio. The absence of bound Xe signal is anticipated due to lower exchange compared to Xe relaxation. A long T1 (660 s) observed for this CB[5] system was similar to that of Xe in pure D2O which confirmed no strong superficial binding site existing for Xe with CB[5]. Additionally, subtle differences in gas binding was reported as a function of the methine sites functionalization in CB[5] [Using hp in CB[5] . To promin CB[5] .129Xe such that its binding capabilities and Xe exchange rates contribute to a growing field of Xe-NMR applications. As its internal cavity (5.8 Å) is a bit larger than that of CB[5], Xe (~4.3 Å diameter) experiences transient binding with medium exchange rates on the Xe NMR time scale. The solubility of CB[6] has to be enhanced such that a direct detection using Xe NMR might be feasible in order to derive the thermodynamic and kinetic parameters linked to its encapsulation process. In this context, Dmochowski and co-workers achieved a higher concentration of CB[6] (>10 mM) by dissolving the commercially available CB[6] in a customized phosphate buffer system with pH 7.2. Using hp 129Xe-NMR, it was shown for CB[6] at 5 mM concentration that the bound Xe peak  appeared at 72 ppm upfield shifted from the dissolved Xe peak at 193.5 ppm, respectively. It has to be assumed that the observed Xe chemical shifts are altered due to the presence of monovalent cations at the carbonyl oxygen available at CB[6] portals. The less water soluble CB[6] has received increasing attention over recent years for utilizing it as a host for encapsulating hp 129Xe-NMR exchange spectroscopy (EXSY) was utilized to determine the thermodynamic/kinetic parameters linked to Xe encapsulation by CB[6]. The association constant determined for Xe with CB[6] in PBS was found to be in line with values anticipated for Xe-host interactions   by appending six cyclohexyl (Cy) units at the equatorial positions. This approach tremendously increased the solubility of CB[6] from less than 10−5 M to 2 × 10−1 M albeit without affecting its cavity dimensions and guest binding properties. The binding constant of Xe•Cy6CB[6] was found to be 3.4 ± 0.1 × 103 M−1 (determined by ITC) which is comparable to the binding of Xe to water soluble cryptophanes  was not possible. It was thus also measured in the presence of 0.2 M Na2SO4 and both signals were shifted downfield, presumably due to the interactions between the encapsulated Xe and the cations on the portals  measured either through change in its proton chemical shifts or through another guest competition such as THF, respectively [T1 relaxation time of Xe in water-soluble CB[6] was calculated to be approximately 40 s and it is much larger than those determined for water soluble cryptophanes  even after 64 acquisitions with hp 129Xe. However, the presence of a second pool that is in fast exchange can be seen by an accelerated decay of the acquired FID signal from the dissolved gas and a corresponding free Xe in solution peak at 190 ppm with an increased line width. A signal for Xe CB[7] was proven only through indirect detection, i.e., HyperCEST-based detection  and b) it is possible to apply relative strong saturation pulses without causing a “spillover” effect . Xe is an ideal and versatile nucleus for this technique since it undergoes many transient interactions with molecular partners. Its large chemical shift range enables excellent selectivity when applying the saturation pulse and allows “multiplexing” for working with multiple guests . It is tn]s was originally motivated by identifying a Xe host with a faster exchange in aqueous solution than the one observed for cryptophane-A (CrA). The latter one is a cage-shaped molecule that had been introduced as a building block for Xe biosensors in MRI applications  is actually hard to identify without substantial signal averaging, even for hp 129Xe. Together with the limited solubility of CB[6] in water in the absence of solubilizing cations, the CEST approach is the ideal way to identify the binding of Xe with this host. The z-spectra, however, show a clear response at ca. 100 ppm upfield from the signal of free Xe. The direct comparison with CrA clearly demonstrated the superior HyperCEST performance of CB[6] for imaging applications  only surpasses the achieved CEST effect when B1 is strong enough to achieve sufficient saturation during the much reduced residence time of the Xe spins inside the cavity. This is illustrated in Indeed, the faster exchange of Xe with CB[6] causes already significant line broadening in conventional direct ications . HoweverkBA of Xe from the host (pool B) into the bulk pool (pool A). It is a simple parameter to classify the constant complex association/dissociation of the CEST spin label. The value is derived from quantitative HyperCEST measurements (see −1 for CB[6] but only 11%⋅s−1 for CrA in pure water at room temperature. This is a good example that the sensitivity of HyperCEST clearly benefits from a loose interaction with accelerated exchange (ca. 55-fold faster compared to CrA) despite a ca. 1.7-fold reduced occupancy of the host.This initial HyperCEST MRI study of CB[6] also introduced the gas turnover rate, ents see  and was n] host. Exchange rates of reversibly bound Xe are very susceptible to the cavity size and portal accessibility that the noble gas interacts with [kBA of bound Xe back into the bulk pool. While Xe exchange proved very efficient for CB[6], the rate for CB[5] is too slow to benefit from neither hp nuclei nor an exchange-based signal transfer. Not surprisingly, CB[7] shows the opposite effect and exhibits rather fast exchange. This host was first investigated as an alternative to CB[6] where a higher host concentration was needed due to a large number of competing guests in cell lysate  and larger hosts, the exchange rates are easily in the kHz regime and thus this recommended value for B1 might already be too demanding for cw RF application. The following estimations give an impression about the range of CEST efficiency in cases where reduced powers are necessary:B1 = kBA/γ yields 50 % of maximum depolarization rate possible, namely on-res = (fBkBA)/2;B1 ≤ kBA/γ yields on-res ≅ (fBkBA)(γ B1)2 which is parabolic in B1 for this low power regime.As demonstrated by the direct comparison between CrA and CB[6], the faster exchange of Xe with the latter host requires adapting the saturation conditions. Excessive saturation power should be avoided to preserve sufficient spectral resolution (and for respecting RF power application limits for future in vivo studies) but weak saturation is inefficient in affecting the magnetization during the shortened residence time inside the host. Each system therefore has an optimum saturation power up to which the CEST effect for a constant saturation time increases to a notable extent. Further increase of B1, the full width at half-maximum of the CEST response is given by B1 ≥ kBA/γ shows a linear dependence Γ ≅ 2γB1;relative strong saturation with B1 ≤ kBA/γ yields Γ ≅ 2kBA, which is the minimum possible width that is governed by the Xe exchange rate.weaker saturation with When spectral resolution becomes more important due to nearby chemical shifts of exchange-connected pools, one should also consider the width of the signals in the z-spectrum. For a given saturation power on-res and Γ increase with B1, it will be beneficial to evaluate the ratio of fastest depolarization  and width, on-res/Γ, as a function of B1. This ratio illustrates that there is an optimum condition for an intense but reasonably narrow signal. This is reached for a saturation pulse strength corresponding to B1~kBA/γ [As both 1~2kBA/γ .129Xe is not helpful any more to resolve a separate saturation response. Pillar[n]arenes are an example of hosts that have been recently demonstrated to form box-shaped structures urils . The lat129Xe HyperCEST and 19F GEST NMR allow a quantitative analysis of z-spectra to obtain absolute values for various parameters that characterize the 2-pool system. The analysis relies on solving the Bloch-McConnel equations for exchanging spin pools under the influence of a saturation pulse. Details will be discussed in the following section. Both Although the HyperCEST detection sensitivity can reach picomolar concentration ranges, a z-spectrum per se does not provide direct quantitative information about the inclusion complexes. An important aspect of z-spectra is that the intensity of the CEST response does not scale linearly with the host concentration. At high concentrations and/or sufficiently strong saturation pulses, the system quickly reaches a state where the host is re-loaded with already saturated magnetization. This is called “back exchange” and becomes increasingly important when exceeding CEST effects of more than 30%. The theoretical shape of a HyperCEST spectrum was predicted by Zaiss et al. . A set ofB), the Xe release rate (kBA), the chemical shifts of free and bound Xe , the Xe association or binding constant (KA) and the Xe host occupancy (β), respectively. The first test case was the cage-like molecule CrA-ma (11 µM) in H2O with KA = 850 ± 250 M−1; β = 29 % and kBA = 38 ± 6 s−1, implying that CryA-ma is in the slow exchange regime on the NMR time scale s with their relatively fast Xe exchange in the absence of cations. qHyperCEST was applied to CB[6] in H2O where a direct 129Xe-NMR spectrum of CB[6] at 3.4 µM without salt addition yields no signal for bound Xe. Contrarily, using HyperCEST at varying saturation conditions revealed a CEST peak (−95.6 pppm) indicating Xe CB[6] inclusion complexation. qHyperCEST analysis returned the following values: kBA = 2100 ± 300 s−1, KA = 2500 ± 400 M−1, and β = 49%. This was in agreement with the values reported for water-soluble CB[6] derivatives. Comparing the Xe-CB[6] system to Xe-CryA-ma indicated that CB[6] is in an intermediate exchange regime on the NMR time scale. As mentioned in β⋅kBA from qHyperCEST analysis and is the maximum depolarization rate per host molecule for a given Xe concentration. One of the aims of using qHyperCEST is to determine the Xe exchange and binding parameters for conditions that are below the detection limit of directly detected It should be mentioned that exchange-connected pools of Xe can also be investigated beyond a saturation transfer experiment. The common aspect of such detection schemes is manipulation of one pool, followed by observation of the system during equilibration of this perturbation. This has been demonstrated with different implementations for CrA  but alsoThe concept of displacement assays has been widely used for supramolecular systems with competing guests. As an example, supramolecular tandem assays (STAs) based on fluorescence detection have gained much attention due to their label-free approach involving less or no further preparation/separation steps. As described in the introduction, STAs for optical detection involve reporter pairs comprising a macrocyclic host and a fluorescent dye where the binding of the latter one to the host is being suppressed by the stronger binding partner. The reaction course is thus monitored through the change in fluorescence upon displacing the dye. Such STAs can serve as a simple, inexpensive, sensitive, and label-free method for continuous monitoring of different enzymatic reactions . Althoug129Xe HyperCEST NMR  imposes challenges under more realistic conditions that include ions or other guests that interact with the portals or the cavity, respectively. Competing guests in, e.g., cell lysate are already outcompeting Xe at the maximum achievable concentrations of CB[6]. The assay was thus implemented with CB[7] that can be dissolved at higher concentrations. However, the accelerated exchange of Xe yields a rather inefficient CEST effect under these conditions and the z-spectrum is characterized by a broad direct saturation response for unbound Xe. In fact, the origin of the weak CEST response with regard to the broad direct saturation response has been revisited recently  see Sec. A stronacc]) as a function of time. Based on the fast dissociation of Xe from the host and efficient association of any newly produced Cad with CB[6], several assumptions can be made that simplify the qHyperCEST evaluation. The experimental results for probing a 6 mM reservoir of Lys with 16 µM of CB6 confirmed the linear progression of the reaction as only the conversion of the initial 2.6‰ of the substrate has been monitored. The overall reaction velocity depends on the enzyme concentration (see A follow-up study with CB[6] (and no lysate) was performed in a time-resolved approach where the chemical shift information is encoded with the UFC method see  in the ption see a and manT2 relaxation time of 129Xe. Formation of the Cad CB[6] inclusion complex prevents Xe from experiencing frequent changes in resonance frequency. As long as many CB[6] cavities are accessible, the large chemical shift range of Xe induces a rapid loss of phase coherence for nuclei that frequently swap between two different environments. This represents an alternative detection method of displacement assays. The concept of so-called T2ex agents is also known in 1H-NMR from paramagnetic chelates that transiently coordinate H2O  which has an affinity for phenylalanine (Phe). The interaction between this amino acid and the molecular container was first described for insulin  and the 129Xe-NMR. This concept mainly relies on a two-faced guest (TFG) that binds with intermediate affinity to CB[6], but also with its opposite face at higher affinity to a protein target of interest. To build such TFG, butylamine was chosen as it displays intermediate affinity for CB[6] (KA = 2.85 × 105 M−1 in PBS at 300 K) in addition to carbonic anhydrase II (CAII EC4.2.1.1) binding moiety p-benzenesulfonamide. Both building blocks were tethered together in order to generate four TFGs with varying length and chemical structure of the linker. CAII was chosen in this study since it is a model enzyme of biomedical relevance . A special implementation of the displacement concept can be achieved with molecular guests that have two different binding motifs for engaging with different hosts. As such, this method to control and monitor the sequential interaction of different molecules in solution is of general interest as it assists the generation of molecules with useful properties. Applications include, e.g., enzyme- and small-molecule mediated tandem reactions, as well as drug delivery and nanoscale architectures ,170,171.elevance  with a selevance . The actg moiety . The ITCp-nitrophenyl acetate (pNPA)) by absorption measurements in solution. The TFG with higher affinity also exhibited a stronger CAII inhibition. Once complexed to CB[6], the TFG inhibited the CAII only slightly less than TFG alone, thus confirming that the TFG shuttles between CB[6] and CAII. This shuttling is thermodynamically controlled as evidenced through titration of CB[6] with CAII, respectively. This molecular relay concept was extended to NMR in order to generate a “turn-on” strategy-based Xe NMR biosensor. 129Xe-NMR of CB[6] (1 µM) in PBS revealed two peaks at 193 ppm (129Xeaq) and 122 ppm (Xe•CB[6]), respectively. Addition of TFG (4 µM) reduced the bound Xe CEST response at 122 ppm owing to the intermediate TFG binding to CB[6]. Introducing CAII (4 µM) into the solution almost restored the peak at 122 ppm, thus demonstrating that the TFG was sequestered by CAII addition. The molecular relay was also implemented using commercially available pentylamine biotin (pAB) as TFG that binds CB[6] less tightly (KA = 4240 M−1) than its target protein avidin. Addition of 50 µM commercial TFG to CB[6] (1 µM) fully ‘turned off’ the Xe@CB[6] peak, while it was completely ‘turned on’ upon addition of avidin (50 µM), thus conforming the reprogramming of the CB[6]-TFG relay for assaying a wide range of biomolecules in solution. Extending the relay to cellular environments  surprisingly indicated that CB[6] (16 µM) gave a Xe•CB[6] signal in spite of CB[6] experiencing non-specific binding from different components of bacterial cell lysates (as reported in [E.Coli) greatly reduced the CEST response from 16 µM CB[6] compared to lysate from CAII-overexpressing E. Coli in which the original CEST signal intensity was preserved. These findings show the ability of CB[6]-based detection in identifying a specific protein within a complex mixture -TFG complex was incubated with CAII for 20 min while monitoring the hydrolysis rate of the substrate (orted in ). Subseq mixture . 129Xe HyperCEST was utilized to monitor the kinetics of the molecular relay-mediated transition occurring for AMADA-Putr between its complexed state in CB[6] and CB[7]. The CB[6] (4 µM) accessibility for Xe was blocked initially by providing AMADA-Putr (4 µM) leading to a complete loss of the CEST response for Xe@CB[6]. Subsequent addition of CB[7] (5 µM) to the above inclusion complex in solution resulted in an immediate CEST response pertaining to Xe@CB[6]. The course of the reaction indicates that CB[6] was released over time from the ternary complex such that the former is available for inclusion complexation with Xe. Even though CB[6] binds Xe after its release from ternary complex, its CEST signal is not completely recovered due to a remaining small fraction of the ternary complex  solubility in solution can be somewhat enhanced. The molecular ‘threads’ involved in rotaxane formation are basically designed by considering the molecular recognition properties of CB[6], e.g., an affinity for positively charged guests. Such strategy was employed to generate a chemically activated CB[6]-rotaxane platform for ‘turn on-off’ 129Xe biosensors [129Xe accessibility to CB[6] in a ‘turn-off’ state. Inducing cleavage at the respective site in the ‘thread’ resulted in a ‘turn-on’ state of the rotaxane in which CB[6] was freed to facilitate 129Xe binding. The stoppers such as pyrene-functionalized 2-azidoethylamine (PyAA+) and an adamantyl-ester functionalized propargylamine (AdPA+) were linked together by CB[6]-catalyzed azide-alkyne 1,3-dipolar cycloaddition. Usage of β-cyclodextrin as caps was necessary for solubilizing the two stoppers in aqueous solution [129Xe NMR of the fully assembled rotaxane (100 µM) showed no dedicated Xe@CB[6] signal, thus suggesting that CB[6] remains in the ‘turn-off’ state. The rotaxane was ‘turned-on’ through base-catalysed ester hydrolysis  after which a semi-rotaxane was formed as a product. This semi-rotaxane displayed suitable Xe binding and exchange kinetics for producing a HyperCEST response.Regarding rotaxane generation, CB[6] was utilized extensively as the preferred host for accommodating the positively charged ‘thread’ component. Additionally, CB[6] entertains hp osensors . In thissolution ,182,183.129Xe HyperCEST NMR. In this approach, one stopper (PyAA+) was conjugated to -carboxytetramethylrhodamine (TAMRA) as the complementary stopper via an axle comprising a PLG-LAG peptide sequence. The latter is recognizable by matrix metalloprotease-2 (MMP-2). CB[6]-catalyzed azide-alkyne 1,3-dipolar cycloaddition was helpful for installing the CB[6] onto the axle. The ‘turn-off’ state was proven by observing no significant Xe NMR signal for the full rotaxane, thus suggesting that the stoppers remain in place and keep every unit intact. The ‘turn-on’ state of the rotaxane at 5 µM concentration was attained after cleaving it for 24 h by MMP-2 (5 nM). The cleavage led to CB[6] release from the rotaxane axle as revealed by the appearance of a CEST response from Xe•CB[6] (15% CEST effect)  with KA = 5.5 × 102 M−1) and an aryl boronic acid group (a H2O2 cleavable cap), respectively. A non-responsive control rotaxane was generated by tethering it either to a fluorophore or maleimide to facilitate fluorescence measurements and/or for further functionalization to biomolecules of interest. The ‘turn-on’ state of the rotaxane (25 µM) was achieved after cleavage (1 h) initiated through addition of H2O2 (50 µM), thereby leading to an apparent maximum saturation transfer of about 25% from Xe•CB[6]. Mass spectrometry indicated that H2O2 does not influence the structural integrity of CB[6] and the cleaved axle. As a proof of concept for rotaxane functionalization, maleimide was linked to cysteine residues available on the vascular cell adhesion molecule (VCAM)-1 binding peptide or to a tobacco mosaic virus (TMV) protein-based nanoparticle. This protein assembly could be advantageous for drug delivery and imaging applications -based rotaxane approach was also used for studying extracellular Hd states ,186. Theications ,188. VCAoduction ,190. To CEST NMR ,192. p-tert-butylcalix[4]arene , dibenzo-18-crown-6 or [4]arene .The func[4]arene . Another[4]arene . Such in[4]arene .129Xe NMR remains to be done but could give meaningful insights into the cargo release. Likewise, a spontaneous self-assembly of two unsubstituted hosts in water without any elaborative synthesis or derivatization of hosts would also be of interest. Recently, it was shown that CB[7] instantaneously self-assembles upon the addition of water soluble 4-sulfocalix4]arene (SC[4]A) leading to the formation of microcrystals in near-quantitative yield (>90%) in water . This su® (sulfobutylether derivatized β-CD (SBE-β-CD or CS)) to solubilize the less soluble CB[6] in water . This enhanced solubility was suggested to happen through the external interactions occurring between CB[6] and CS promoted through ‘supramolecular-based positive cooperativity’. Such systems are generally studied using 1H and diffusion NMR at higher concentrations (mM range). Conversely, we have studied such systems with 129Xe HyperCEST by relying on the intrinsic molecular motifs of a host for complexing its counterpart. CS with different degrees of substitution (DS) was used, e.g., CS DS6 and CS DS4 within the concentration range of 20–1000 µM for evaluating its impact on the Xe•CB[6] signal arising from the ternary complex formation (Xe•CB[6]-CS). Measuring CB[6] (5 µM) in the presence of CS DS6 (100 µM) in aqueous solution revealed Xe•CB[6] signal with an intensity reduction and peak shift compared to Xe•CB[6] alone. Additionally, a linear change in Xe•CB[6] peak shift was observed with increasing CS DS6 concentration. In order to achieve a similar change in the observed chemical shift of Xe•CB[6] peak, higher concentrations of CS with the lower DS4 are required. This differently pronounced degree of interaction is thus attributed to the presence of different DS available on the CS rims. The specific molecular recognition pattern of CB[6] (5 µM) for SBE chains available on CS  was checked by competitively inhibiting the latter by structurally similar butylsulfonate (BS) at different concentrations (100–1000 µM), respectively. The Xe•CB[6] peak of BS-inhibited ternary complexes was noted at −91.09 ppm compared to CB[6] plus CS DS6 (−92.67 ppm) and CB[6] alone (−95.66 ppm), respectively. Our investigations reveal that indeed the SBE chains on CS are essential for facilitating ternary complexation with CB[6] and 129Xe probes such spontaneous self-assembly at relatively low concentration (µM regime). Such 129Xe HyperCEST-based studies might pave way for studying multiple interactions occurring within complex supramolecular structures, e.g., capsules, machines, necklaces etc. at ease s structure can lead to the generation of hosts with intriguing properties for different applications such as molecular imaging or targeted drug delivery. Due to high water solubility, mono-functionalization of CB[7] is preferred over modification of the “even” members of the CB[n] family]. However, it is a challenging task to introduce functional groups on CB[n] skeletons  [n]s [n] finds interesting applications in supramolecular chemistry and drug delivery [n]s with HyperCEST can thus provide insights into the cargo stability and release. Introduction of a functionalizable handle on the CB  can be sed CB[6]  and/or m[6] [n]s . Despitedelivery . Investin] has investigated the option to detect a specific protein target in solution using 129Xe HyperCEST. A biotinylated CB[7] (btCB[7]) was utilized for this work. Originally, mono-functionalized btCB[7] was synthesized as a container molecule for delivering anti-cancer drugs to murine lymphocytic leukemia cancer cells expressing higher levels of biotin receptors [129Xe HyperCEST of control CB[7], i.e., unmodified CB[7], indicated a response in agreement with previous reports [iCB[7] [iCB[7] possesses a reduced internal cavity diameter (~5.5 Å) due to the inverted flip of a glycoluril unit in its structure. It was hypothesized that a signal from CB[7]-bound Xe is presumably too broad for well resolved CEST detection. However, the existence of Xe•CB[7] manifests in the dissolved Xe peak with broad saturation response [ibtCB[7] response is significantly weaker than the Xe•iCB[7], possibly due to less ibtCB[7] formed during synthesis compared to iCB[7]. One might initially not exclude other inter- and intra-molecular interactions that might have played a role in observing a reduced response for Xe•ibtCB[7], e.g., exclusion complexation between ibtCB[7] and its own biotin tail or with that of a neighboring ibtCB[7]. However, the biotinylated tail interaction with CB[7] portals or the cavity has been excluded through MD simulations-based results. Another option might be that ibtCB[7] dimerizes in solution. Indeed, dimer formation was supported through MALDI and MD simulation-based results. However, the strong avidin binding to biotin is sufficient enough to hamper the btCB[7] dimerization [n]. Recently, the first study with a mono-substituted CB[eceptors . The 129 reports . The Xe• [iCB[7] . The iCBresponse . The Xe•rization  anticipaibtCB[7]), 0 ppm (free Xe) and additional new signals at δ = −40 and 100 ppm, respectively. The resonance at 100 ppm was ascribed to a direct interaction of Xe atoms with avidin. The significant response at δ = −40 ppm was attributed to Xe inside btCB[7] bound to avidin (Xe•btCB[7]-avidin). This was proven by observing no signal in the case of unmodified CB[7] in the presence of avidin. The same signal was also missing for btCB[7] in the presence of avidin that was pre-saturated with biotin. Such interaction between btCB[7] and avidin was estimated to generate ~50% CEST difference adequate for achieving a significant image contrast. Thus, introducing these hosts in close proximity to protein targets generates functionalized CB[n]-based biosensors for different imaging applications [The btCB[7] (50 µM) treated with avidin indicated four distinct signals, namely −68 ppm  [IC50 value of 0.53 mM was determined. In vivo studies revealed a maximum tolerated dosage of 250 mg kg−1 for CB[7] in mice. The toxicology has also been investigated in [n] family  with results confirming a very low toxicity of these nanocontainers. Cellular uptake was followed by intracellular release of container-loaded drugs. Altogether, these toxicity profiles encourage further studies with CB[7] as the one with preferable water solubility for medicinal and pharmaceutical use. Xe itself is a harmless noble gas. It passes the blood brain barrier and easily penetrates into cells. The only potential side effect comes from its anesthetic impact [n]s and related compounds, Xe NMR/MRI could make an important contribution to comprehensive therapeutic and diagnostic approaches. Regarding the biocompatibility of such systems for biomedical applications, some cell culture and n = 7,8) . They ingated in  for fivec impact ,217 but c impact . Overallc impact  where itc impact . The appc impact . Furtherc impact ,223,224 c impact  has beco


Scientific Reports 10.1038/s41598-020-59538-7, published online 13 February 2020Correction to: This Article contains a typographical error in the Site Description section.“According to local meteorological records from 2011 to 2018, the main winds direction is northeast and north .”should read:“According to local meteorological records from 2013 to 2017, the main winds direction is easterly and westerly .”−1) are incorrect. The correct Figure 2 appears below as Figure Additionally, this Article contains errors in Figure 2. In Figure 2, the ranges of wind speed m.s− are incoAs a result, the Figure legend,“Wind rose at northeastern margin region of Ulan Buh Desert from 2011 to 2018.”should read:“Wind rose at northeastern margin region of Ulan Buh Desert from 2013 to 2017”


Peroxisome proliferator-activated receptor gamma (PPARγ), a member of the nuclear receptor superfamily, participates in multiple physiological and pathological processes. Extensive studies have revealed the relationship between PPARγ and various tumors. However, the expression and function of PPARγ in bladder cancer seem to be controversial. It has been demonstrated that PPARγ affects the occurrence and progression of bladder cancer by regulating proliferation, apoptosis, metastasis, and reactive oxygen species (ROS) and lipid metabolism, probably through PPARγ-SIRT1 feedback loops, the PI3K-Akt signaling pathway, and the WNT/β-catenin signaling pathway. Considering the frequent relapses after chemotherapy, some researchers have focused on the relationship between PPARγ and chemotherapy sensitivity in bladder cancer. Moreover, the feasibility of PPARγ ligands as potential therapeutic targets for bladder cancer has been uncovered. Taken together, this review summarizes the relevant literature and our findings to explore the complicated role and function of PPARγ in bladder cancer. Bladder cancer (BCa) is one of the most prevalent malignant tumors in the urinary system Previous studies have compared bladder cancer with paracancerous tissues, and showed the controversial consequences of the expression of PPARγ in bladder cancer. Cheng et al. searched the Oncomine database and found that there was amplified mRNA expression of PPARγ in BCa tissues Gene ontology (GO) enrichment analysis of BCa datasets from The Cancer Genome Atlas indicated that PPARγ was related to the regulation of cell proliferation, apoptosis and enhancement of biosynthetic processes Ligand binding activates PPARs to translocate to the DNA and bind to retinoid X receptors (RXRs) to form heterodimers and then bind to specific PPAR response elements (PPREs) PPARγ inhibits SIRT1 at both the transcriptional and translational levels after directly binding to the SIRT1 promoter As early as 15 years ago, it was shown that the variant genotype of PPARγ indicated a lower recurrence risk among untreated patients with BCa, and MIBC exhibited a significantly higher frequency of variant PPARγ genotypes than NMIBC These results revealed that the point mutation that activated PPARγ or RXRα mutation that acted through PPARγ-dependent pathways The role of ROS metabolism in cancer biology is complicated, similar to a double-edged sword, and it regulates both cell survival and apoptosis, depending on the dose, duration, type, or location The results of our group revealed that when SIRT1 was knocked down in BCa cells, ROS production was reduced, accompanied by an increase in antioxidant enzymes and total/acetylated FOXO3a, a transcription factor that is the most closely connected to SIRT1 among the FOXO family Reprogramming energy metabolism has been recognized as a hallmark of cancer Our group has performed microarray analysis with human bladder samples. The results revealed that the PPAR signaling pathway mattered greatly in fatty acid/lipid metabolism in bladder cancer Of bladder tumors, nearly 70% are low-grade non-muscle invasive cancers, which are not life-threatening but have a distinct tendency to relapse. Maintaining life-long surveillance is very expensive In addition, presurgical (neoadjuvant) cisplatin-based chemotherapy (NAC) is the preferred therapy for high-risk MIBC Many reports have suggested that the regulation of agonists or antagonists on PPARs in cancer cells may be a potential therapeutic strategy for metabolic diseases and cancers, including bladder cancer Concerns arise when considering PPARγ ligands as potential chemopreventive and therapeutic agents for bladder cancer. Researchers found that PPARγ agonists cause bad results in rats, not only in bladder cancer PPARγ is a member of the PPAR family and is expressed in a variety of cancers Abnormal metabolism is an important feature of malignant tumors Other studies have hypothesized that the PTEN and PI3K-Akt signaling pathways may be important mechanisms for the downstream regulation of PPARγ. Moreover, complex interactions between PPARγ and other molecules, such as negative feedback loops with SIRT1, WNT/β-catenin, and TGF-β1 and reciprocal regulation with Nrf2, also exist in a variety of physiological and pathological conditions, but the specific mechanism still needs to be further investigated.PPARγ is a ligand-activated transcription factor. Previous studies have shown that PPARγ receptor agonists inhibit the proliferation and growth of tumor cells and enhance the sensitivity of tumor cells to chemotherapies in other malignant tumors. Unfortunately, few studies have investigated the synergistic effects of PPARγ agonists and chemotherapy drugs, such as gemcitabine and docetaxel, in bladder cancer. Taken together, basic research and clinical trials are needed to determine whether PPARγ is a promising biomarker for bladder cancer diagnosis and an effective target for bladder cancer treatment.


The “A-a” oxygen gradient was higher on PAV and baseline ventilation than on NAVA (20.8 (SD 12.3) and 22.9 (SD 11.8) versus 18.5 (SD 10.8) kPa, p = 0.015, < 0.001, respectively).Both proportional assist ventilation (PAV) and neurally adjusted ventilatory assist (NAVA) provide pressure support synchronised throughout the respiratory cycle proportional to the patient’s respiratory demand. Our aim was to compare the effect of these two modes on oxygenation in infants with evolving or established bronchopulmonary dysplasia. Two-hour periods of PAV and NAVA were delivered in random order to 18 infants born less than 32 weeks of gestation. Quasi oxygenation indices (“OI”) and alveolar-arterial (“A-a”) oxygen gradients at the end of each period on PAV, NAVA and baseline ventilation were calculated using capillary blood samples. The mean “OI” was not significantly different on PAV compared to NAVA (7.8 (standard deviation (SD) 3.2) versus 8.1 (SD 3.4), respectively, Conclusion: Both NAVA and PAV improved oxygenation compared to conventional ventilation. There was no significant difference in the mean “OI” between the two modes, but the mean “A-a” gradient was better on NAVA. Mechanical ventilation can be live-saving in the neonatal period but is also associated with complications such as bronchopulmonary dysplasia (BPD) and chronic respiratory morbidity . Modes oThe results of short-term studies –15 suggeclinicaltrials.gov with the identifier NCT02967549. Written informed parental consent was obtained.Infants were recruited from the tertiary neonatal unit at King’s College Hospital NHS Foundation Trust between June 2017 and July 2018. Infants were eligible for the study if they were born at a gestational age of less than 32 weeks and remained ventilated at or beyond 1 week after birth, that is, they had evolving or established BPD [Infants were routinely ventilated via shouldered Coles endotracheal tubes  which have been shown to have minimal to no leak . Prior tNAVA was delivered by the Maquet Servo-n ventilator. A six French Edi catheter of appropriate length for the infant’s weight  was inserted oro- or nasogastrically prior to the commencement of the study and left in place until the study was completed. The Edi catheter was positioned as per the manufacturer’s guidance using the specialised tape measure in the packaging and correct position confirmed using the Edi catheter positioning guide function on the ventilator . The Edi was monitored using the Servo-n ventilator throughout the entire study, including on baseline settings, and whilst PAV was delivered via the Stephanie ventilator.2O above that set on baseline settings, as the Servo-n ventilator will not deliver pressures more than 5cmH2O below this limit. Ventilator parameters were manually collected with a single value that was expected to represent the existing stable baseline.During NAVA, the NAVA level was set by observing the displayed pressure waveform on the ventilator during three to five breaths, whilst the infant was ventilated using their baseline settings and then adjusted so that the pressure on NAVA closely matched that delivered on baseline settings, aiming for a peak Edi of between 5 and 15 μV. The apnoea time was set to 2 s to avoid hypoventilation and the Edi trigger to 0.5 μV as recommended by the manufacturer for neonatal use. The positive end-expiratory pressure (PEEP) was set at the same level as during baseline ventilation. Maximum PIP was initially limited to 5cmH2O [PAV was delivered using the Stephanie ventilator . Whilst ventilating on baseline settings, the infant’s compliance was recorded from the ventilator display every 10 min. The mean of the six results was calculated to determine the degree of elastic unloading required to return the infant’s compliance to normal, that is, 2 ml/cmH2O . Initial2O . No resi2O , 24. The2 was adjusted to maintain saturations between 92 and 96% as per the Unit’s guidelines. Expiratory tidal volume, FiO2, respiratory rate, MAP, PIP and PEEP were recorded from the ventilator display (no PIP or PEEP is displayed during PAV). Blood gas analysis was performed using capillary samples. Capillary samples were not formally arterialised. Blood gas samples were analysed immediately using the blood gas analyser on the neonatal unit .The FiO2*100)/. Secondary outcomes included the “A-a” gradient calculated as (FiO2*(Patm – pH2O) – (PCO2/0.8)-pO2), where Patm = 101.33 kPa and pH2O = 6.3 kPa; the oxygen saturation to fraction of inspired oxygen ratio (SF ratio) calculated as SpO2/FiO2 and the PO2/FiO2 ratio. In addition, the peak and tonic Edi during each epoch were obtained from the Maquet ventilator.As capillary blood samples were used, we calculated quasi OIs and alveolar-arterial oxygen gradients which we have designated “OIs” and “A-a” gradient, respectively. The primary outcome, “OI”, was calculated as  between the results on the two modes.A sample size of 18 infants was chosen as this would allow detection of one standardised difference in the results with 80% power at the 5% significance level. In a previous study , we repoResults were compared from the end of each period of PAV, NAVA and conventional ventilation. Data were assessed for normality using a Shapiro-Wilk test. Normally distributed data were analysed using a one-way repeated measures ANOVA with Bonferroni correction. If the assumption of sphericity was violated, a Greenhouse-Geisser correction was applied. Non-normally distributed data were analysed using a Friedman test. Statistical analysis was carried out using IBM SPSS Statistics version 25 Eighteen infants were studied Table  eight m. NAVA lep = 0.7), but on both PAV and NAVA, the mean “OI” was significantly lower than on baseline ventilation (Table 2 was higher on PAV than on NAVA (mean 0.39 (SD 0.15) versus 0.37 (SD 0.13) p = 0.024) and the MAP lower on PAV than on NAVA (8.3 cmH2O (SD 1.1) versus 9.0 cmH2O (SD (0.9), p = 0.025)) , but the “OI” was not significantly different on PAV and NAVA (p = 0.29).There was no significant difference in the mean “OI” at the end of 2 h on PAV compared to on NAVA (mean 7.8 (standard deviation (SD) 3.2) versus 8.1 (SD 3.4), p = 0.015.) It was also significantly lower on NAVA than at baseline , but there was no statistically significant difference between PAV and baseline  , but there was no statistically significant difference between the results on PAV and NAVA (p = 0.18). The SpO2/FiO2 ratio was lower on PAV and baseline than on NAVA (p = 0.004) . The respiratory rate was significantly higher on PAV than on NAVA. The peak and minimum Edi were not significantly different between modes of ventilation . The PO2/FiO2 was lower on PAV, but this did not reach statistical significance.We have demonstrated that there was no significant difference in mean “OI” between PAV and NAVA, but both modes resulted in a reduction in “OI” compared to baseline conventional ventilation. We used the “OI” as the primary outcome as this gives a summary of the effectiveness of each ventilator mode compared to baseline. The mean airway pressure, however, was lower and the FiOer delay –19. The 2 and PIP [p = 0.21). There was also no difference in the incidence of BPD, but the study was not powered to detect this [Both PAV and NAVA have been compared to conventional and other triggered modes of ventilation in small short-term studies in infants. PAV as compared to pressure-controlled conventional ventilation has been shown to reduce the OI in infants with respiratory distress syndrome  and to r and PIP . There hect this .2 was statistically significantly higher on PAV than on NAVA, as was also found in our study. The small increase in PaCO2 may be due to the way that NAVA and PAV switch to back up ventilation after an apnoea period or to the amount of unloading during PAV and the NAVA level set. If either were to be used for longer periods, then careful monitoring and adjustment of settings as required would be advised.To our knowledge, there have been no previous studies in the neonatal population that have compared NAVA and PAV, but a study in adults compared NAVA and PAV to baseline pressure support ventilation . The OI There are strengths and some limitations to our study. We calculated the “OI” and “A-a” gradients from capillary samples, but this was a crossover study, so each infant acted as their own control. Thus, whilst arterial samples may have been more precise, we suggest that use of capillary blood samples would not have substantially influenced our results. We did not use arterialisation of the capillary samples as there is controversy in the literature as to whether this improves the correlations with arterial results. Furthermore, capillary blood gases have been shown to correlate well with arterial gases if infants are stable and not cardiovascularly compromised , particuThe NAVA level was set as per the manufacturer’s guidelines. Using the manufacturer’s guidelines, the MAP level during NAVA was decreased compared to baseline setting suggesting that the level was appropriate. All of the infants in our study either had or were subsequently diagnosed with BPD, indicating that our inclusion criteria successfully identified those with evolving or established BPD. The wide range of postnatal ages in our study population may have influenced our results, but advantages of PAV and NAVA over conventional ventilation have been demonstrated in infants with a variety of postnatal ages in previous studies. Targeted tidal volumes of between 5 and 7 mls/kg were used, as studies have shown chronically ventilated infants may require 7 mls/kg , 30 and In conclusion, we have found no significant differences in the “OI” after 2 h of support by PAV or NAVA but both significantly improved the OI compared to baseline ventilation. Other indices, as the ‘A-a’ gradient and SF ratio demonstrated that NAVA improved oxygenation over PAV, but the differences were relatively small.


However, no relation has been found between whitefly density and CBSD incidence and severity on cassava leaf, root and stems. Molecular analysis showed the incidence of single infections of UCBSV was greater than single infections of CBSV and mixed infections of UCBSV and CBSV. Disease incidence was greater in Ituri than in Haut Uélé; molecular incidence was lower than field incidence. Our results raise the need for appropriate CBSD control strategies in DRC.Cassava fields were prospected from two provinces of the Democratic Republic of Congo (Ituri and Haut Uélé) to evaluate the ampleness of Cassava brown streak disease (CBSD) infection. CBSD pressure was determined by assessing the incidence, severity, whitefly abundance and distribution of the disease viruses in the surveyed provinces. A duplex RT-PCR was performed for the simultaneous detection of Ugandan Cassava brown streak virus (UCBSV) and Cassava brown streak virus (CBSV) on 56 cassava leaves sampled in the study area. Our results show a high field CBSD incidence contrasted to a low severity in both provinces. CBSD severity was similar in both provinces (mean disease severity 2). High densities of whitefly were recorded in Ituri province (10 adult whiteflies plant In each field, we assessed leaf CBSD symptoms in 30 × 3–10 months-old plants of the dominant cultivated variety along two transects that extended diagonally from opposing corners of the field and intersected at the field-center.Foliar symptoms Figure ) were clTen plants (>10 months-old) were selected at random along the two transects and we assessed their roots for constriction Figure ) and necWe collected 56 leaf samples, such symptomatic and asymptomatic third fully expanded leaf material from plant for virus analysis at NaCRRI-Uganda Laboratory (42 leaves were collected from Ituri and 14 from Haut Uélé). These leaf samples were pressed between pieces of newspaper and placed in a herbarium press, and stored at room temperature prior to molecular analysis.Disease incidence was calculated as the proportion of sampled plants (percent) that showed disease symptoms, and scores for symptom-free plants were omitted in the calculation of mean incidence and severity for each cultivar and location  on the 5 apical leaves of each of the 30 plants per field that had been assessed for disease symptoms was recorded.Abundance of the principal vector  according to the manufacturer’s instructions. The extracted cDNA was amplified using specific primers (CBSDDF2 [Fwd 5ʹ-GCTMGAAATGCYGGRTAYACAA-3ʹ] 437 bp UCBSV and CBSDDR [Rev 5ʹ-GGATATGGAGAAAGRKCTCC-3ʹ] 343 bp CBSV), as described by Mbanzibwa et al. , for the3.3.1.Incidence of CBSD in cassava leaf and tuber material was greater in Haut Uélé  than in Ituri  (Table 3.2.Mean CBSD severity was similar in Haut Uélé and Ituri (severity index: 2) Table . CBSD syIn Haut Uélé, five of the eight infected cultivars showed symptoms on leaf material (5/8), one showed brown streaks on the stem (1/8), and three exhibited necrosis in the roots (3/8). Our results show that there were positive associations between root incidence and root severity , leaf incidence and leaf severity , leaf severity and stem severity , CBSD root severity and stem severity   on leaves, stems, and tuberous roots. However, most plants tended to show symptoms on either leaves or roots Table . These rSimilar visible levels of severity have been observed by Bigirimana et al.  in areasThe positive associations between disease incidence and severity in leaf and root material, between disease severity in leaf and stem material, and between disease severity in the root and stem material indicate that severity of CBSD affects tuber health. Similar case was found in western Kenya, where Masinde et al.  reported−1 recorded in Ituri) compared in other CBSD outbreak areas, such as the African Great Lakes region, where Bigirimana et al.  in the cultivation zones  than in Haut Uélé (14.29%) Table . These lIn general, we found a wide distribution of CBSD across cassava growing areas in northeastern DRC, highlighting the possibility that other low-altitude areas in central Africa, which were previously thought to be unaffected by CBSD, may see increases in prevalence in CBSD. Mohammed et al.  associatThe high levels of CBSD incidence recorded from fields in Haut Uélé (55.6%) and in Ituri (28%) may be attributed to the planting of infected cuttings sourced from the farming community (Legg et al., Our study supports the hypothesis that CBSD continues to spread from East to West Africa through countries in central Africa. Confirmation of CBSD in eastern DRC (Casinga et al.,


V˙E) in HFrEF. Ten patients accomplished 4‐min cycling at 60% peak workload and then recovered for 2 min under either: (a) 21% O2 inhalation (tonic carotid chemoreflex activity) with legs’ circulation free (inactive muscle metaboreflex); (b) 100% O2 inhalation (suppressed carotid chemoreflex activity) with legs’ circulation occluded (muscle metaboreflex activation); (c) 21% O2 inhalation (tonic carotid chemoreflex activity) with legs’ circulation occluded (muscle metaboreflex activation); or (d) 100% O2 inhalation (suppressed carotid chemoreflex activity) with legs’ circulation free (inactive muscle metaboreflex) as control. V˙E, tidal volume (VT) and respiratory frequency (fR) were similar between each separated reflex (protocols a and b) and control (protocol d). Calculated sum of separated reflexes effects was similar to control. Oppositely, V˙E  and fR  increased versus control when both reflexes were simultaneously active (protocol c). Therefore, the carotid chemoreflex and the muscle metaboreflex interacted to V˙E regulation in a fR‐dependent manner in patients with HFrEF. If this interaction operates during exercise, it can have some contribution to the HFrEF exercise hyperventilation.Synergism among reflexes probably contributes to exercise hyperventilation in patients with heart failure with reduced ejection fraction (HFrEF). Thus, we investigated whether the carotid chemoreflex and the muscle metaboreflex interact to the regulation of ventilation ( Our results support that the carotid chemoreflex and the muscle metaboreflex interact to the regulation of ventilation in patients with chronic heart failure with reduced ejection fraction (HFrEF), which may have implication for exercise hyperventilation in this population. Furthermore, the effect mediated by separated reflexes was not associated with the effect yielded when both reflexes were simultaneously active. Hence, reflexes interaction should be further explored to advance the understanding of exercise hyperventilation in HFrEF. Subjects were assessed at the Antonio Pedro University Hospital and at the Biomedical Institute of the Fluminense Federal University, in Niteroi, Rio de Janeiro State, Brazil. The Ethics Committee of the Fluminense Federal University approved the study (CEP‐CCM/HUAP 36681414.0.0000.5243), and the study conformed to the standards set by the Declaration of Helsinki. All subjects provided written informed consent to participate in the study.Ten patients (7 men) with HFrEF met the inclusion criteria and volunteered to participate in the study. Eligibility criteria for all were: age between 40 and 70 years, reduced left ventricle ejection fraction (Simpson < 45%), optimal pharmacological treatment, New York Heart Association (NYHA) class II or III, no hospitalization in the last 6 months, body mass index lower than 35 kg/m2.2The experimental protocol encompassed five visits. On visit 1, subjects underwent an incremental workload exercise. On visit 2, subjects were familiarized with cuffs inflation and constant workload exercise. In some cases, the familiarization visit was repeated to guarantee subjects’ adaptation with the protocol. Then, on visits 3 and 4, subjects underwent four constant workload exercise trials. Two trials were conducted per day, with at least 30 min of interval. At last, on visit 5, subjects’ carotid chemoreflex sensitivity was assessed. Subjects were asked to avoid strenuous physical activity and refrain from consuming caffeine for 24 hr before arriving for all visits. All visits were separated by at least 24 hr.2.32.3.1Incremental workload exercise was performed on an electromagnetic cycle ergometer . Workload was progressively increased until volitional fatigue or until pedal cadence (60 rpm) could not be maintained despite strong verbal encouragement  and two circulatory states in the lower limbs (circulatory occlusion and free flow) were combined during the recovery period. A hundred percent of O2 was used in the inspired air in an attempt to provoke a fast and robust suppression of the tonic carotid chemoreceptors activity . Exercise intensity was set at 60% peak workload attained in the incremental workload exercise  100% O2 inhalation with circulatory occlusion in the lower limbs to suppress the tonic carotid chemoreflex activity with muscle metaboreflex activation; (c) 21% O2 inhalation with circulatory occlusion in the lower limbs to have both reflexes simultaneously active; and (d) 100% O2 inhalation with free flow to the lower limbs to suppress the tonic carotid chemoreflex activity without muscle metaboreflex activation, which consequently was used as a control condition. The order of the first condition of a day was selected by randomization, and the opposite condition was used next. For example, if 21% O2 plus free flow was the first sorted condition, it had to be followed by 100% O2 plus circulatory occlusion in the same visit. In the next experimental visit, for the same subject, another randomization was done to select the order of the remaining conditions.In sum, four different conditions were formed Figure : (a) 21%2 or 100% O2) or rebreathing of expired air  to avoid a change in the O2 delivery to skeletal muscles during exercise, which could influence the metabolic response to exercise  at the end‐exercise level, because circulation occlusion traps a large amount of CO2 in the lower limbs. As a result, the circulating level of CO2 reduces, which may depress carotid and central chemoreceptors  manually controlled the amount of fresh gas delivered to achieve the target PETCO2. So that, high PETCO2 prompted increase of fresh air delivery consequently decreasing rebreathing, whereas low PETCO2 prompted decrease of fresh air delivery consequently increasing rebreathing.A breathing circuit was connected to the pneumotachometer at onset of the recovery period allowing either delivery of fresh air  was initiated 5 s before the end of exercise through rapid cuff inflation . One cuff was used per limb. Cuffs were inflated simultaneously to 20 mmHg above peak systolic arterial pressure (SAP) recorded in the arm during the incremental workload exercise . Cardiac electrical activity was measured by a 12‐lead electrocardiogram for clinical purposes . Arterial pressure was measured via the auscultatory method.An oro‐nasal silicone mask  was tightly adjusted to the subjects’ face to avoid air leak. A bidirectional pneumotachometer was connected to the mask  to allow the measurement of 2.3.3V˙E and pulse oxygen saturation (SpO2). Then, subjects inhaled pure nitrogen for two to eight consecutive respiratory cycles. Inhalation of pure nitrogen was repeated six to 10 times. Repetitions were done after V˙E and SpO2 returned to the baseline level, which usually took 2–5 min. SpO2 was measured at a hand finger . Air flow and pulmonary gas exchange were acquired breath‐by‐breath with a metabolic analyzer . The average of the two highest consecutive V˙E values, either during or after each nitrogen inhalation, was plotted against the respective lowest SpO2. Then, a linear regression was performed. The resulting slope was considered as an index of carotid chemoreflex sensitivity of an individual. Values up to 0.675 L/min/% were considered within the normal range , minimal (min) and maximal (max). Data compared by inferential statistics are presented as mean ± standard error of the mean (SEM). All inferential analyses were two‐tailed. Absolute p‐values were reported whenever possible. p‐values were interpreted taking into account the .05 value as an orientation, but not as cutoff, according to accumulating statisticians’ recommendations  + (protocol b − protocol d)]. “Experimental sum” was considered as the change provoked by simultaneous reflexes activities versus control . Changes versus control were considered the main study's endpoints. Thus, any change data located beyond 1.5 times the interquartile range for a variable, under a given condition, was considered as outlier. Raw data were contrasted to control via two‐way ANOVA, without outlier data. Changes versus control were compared among four conditions via one‐way ANOVA, both with and without outlier data. Fisher's post hoc was used after ANOVA, when needed. Changes versus control were contrasted to zero via single mean Student's 3Table 2 . Furthermore, experimental sum was greater than calculated sum for fR. At last, V˙E experimental sum neither correlated with V˙E separated reflexes effects  nor with resting carotid chemosensitivity . Similarly, fR experimental sum neither correlated with fR separated reflexes effects  nor with resting carotid chemosensitivity .Recovery with tonic carotid chemoreflex activity or muscle metaboreflex activation did not change 4V˙E and respiratory pattern versus control. On the other hand, V˙E and fR increased versus control when both reflexes were simultaneously active . Collectively, these results support an interaction between the carotid chemoreflex and the muscle metaboreflex for the regulation of V˙E and respiratory pattern. Another noteworthy finding was that V˙E and fR experimental sums were neither associated with V˙E and fR separated reflexes effects nor with resting carotid chemosensitivity. The lack of association indicates that the effect mediated by separated reflexes did not predict the effect yielded when both reflexes were simultaneously active. Hence, our results suggest that reflexes interaction should be further explored, particularly in patients who present with altered V˙E and respiratory pattern response to exercise, such as those with HFrEF.The main results of the present study were that sum of separated reflexes effects did not change 4.1V˙E, VT and fR versus normoxia when the leg circulation was unobstructed. It is unlikely that the lack of hyperoxia effect was attributed to insufficient inhibition of carotid chemoreceptors. The reason is that data collected in anesthetized cats indicate that 350 mmHg of O2 pressure in the arterial blood (PaO2) provokes a large reduction in the carotid chemoreceptors activity  on the V˙E and a hyperadditive effect on the fR . Breathing with a facemask could have some effect on V˙E and breathing pattern  to provoke greater V˙E response than the sum of V˙E responses to each separated stimulus in healthy young men, in spite of PETCO2 being ~7 mmHg lower during hypoxic posthandgrip ischemia versus normoxic posthandgrip ischemia , patients with HFrEF showed fR 7 cycles/min greater than healthy controls (Smith & Olson, T was similar to controls (Smith & Olson, fR provoked hypocapnia (Smith & Olson, V˙CO2 data indicates that the hypocapnia could have been eliminated if the excessive fR response decreased by 4 cycles/min. Thus, if the interaction between the carotid chemoreflex and muscle metaboreflex contributes to an extra fR increase of 2 cycles/min during exercise, it would represent 50% of the exaggerated fR response to exercise at an iso‐V˙E level.Our results support that the interaction between the carotid chemoreflex and the muscle metaboreflex raises fR‐mediated exercise hyperventilation in patients with HFrEF may include increased dead space V˙E and increased work of breathing (Smith & Olson, The consequences of 5V˙E regulation in a fR‐dependent manner in patients with HFrEF. Therefore, if this interaction operates during exercise, it can have some contribution to the HFrEF exercise hyperventilation.The carotid chemoreflex and the muscle metaboreflex interacted to ACM, LCV, JACT, MLR, HV, ACLN, and BMS designed the project; ACM, EACG, JACT, MLR, and HV collected the data; ACM, JACT, MLR, and BMS analyzed the data; ACM and BMS involved in statistical analyses; ACM, LCV, EACG, JACT, MLR, HV, ACLN, and BMS interpreted the data; ACM and BMS drafted the manuscript; and ACM, LCV, EACG, JACT, MLR, HV, ACLN, and BMS revised the final manuscript.


Gaussia luciferase enzyme robustly detected low affinity interactions and reduced the amount of protein required by several orders of magnitude compared to other reporter enzymes. Importantly, we discovered that this flash-type luciferase exhibited a reaction-induced inhibition that permitted the use of a single protein preparation as both bait and prey thereby halving the number of expression plasmids and recombinant proteins required for screening. This approach was tested against a benchmarked set of quantified extracellular interactions and shown to detect extremely weak interactions (KDs ≥ μM). This method will facilitate large-scale receptor interaction screening and contribute to the goal of mapping networks of cellular communication.Extracellular protein interactions mediated by cell surface receptors are essential for intercellular communication in multicellular organisms. Assays to detect extracellular interactions must account for their often weak binding affinities and also the biochemical challenges in solubilising membrane-embedded receptors in an active form. Methods based on detecting direct binding of soluble recombinant receptor ectodomains have been successful, but genome-scale screening is limited by the usual requirement of producing sufficient amounts of each protein in two different forms, usually a “bait” and “prey”. Here, we show that oligomeric receptor ectodomains coupled to concatenated units of the light-generating  Importantly, these cell surface proteins are targets for therapeutic antibodies that are increasingly used to treat indications such as autoimmunity and cancer2. Despite their medical relevance, however, the specialised biochemical nature of membrane proteins has meant that they are often underrepresented in large-scale protein interaction screens3 which typically employ techniques such as biochemical purifications and transcription-based protein complementation assays4. Biochemically, membrane proteins are difficult to solubilise in their native conformation due to their amphipathic character, and the interactions between cell surface receptor proteins can be extremely transient—having dissociation half-lives of just fractions of a second—making protocols that include stringent wash steps unsuitable to detect them5.Proteins embedded in the cellular membrane are critically placed to transmit signals from the external environment to the interior of the cell to ensure coordinated and appropriate cellular behaviours from the earliest stages of development to the maintenance of adult tissues6) and include mass spectrometry-based techniques for association by proximity labelling8, genetic manipulation of cell surface protein repertoires in the context of a cell membrane11, and expression cloning14. A very successful approach has been the development of ELISA-style assays that detect direct binding events within large libraries of soluble recombinant receptor ectodomains17, including the AVEXIS (AVidity-based EXtracellular Interaction Screening) method developed by our laboratory18. This general approach relies on the ability to express the extracellular binding domain as a secreted recombinant protein that retains the extracellular binding activity, and has been particularly useful for common receptor architectural classes such as type I, type II, and GPI-anchored membrane proteins. It is important that structurally critical posttranslational modifications such as glycosylation and disulfide bonds are retained, and so eukaryotic protein expression systems such as mammalian cells are most commonly used. Usually, the receptor ectodomains are produced in two forms: a “bait” protein that can be immobilised on a solid substrate such as a microtitre plate, and a soluble “prey”. To circumvent the low binding affinities, the prey proteins are purposefully oligomerised to create highly avid binding probes4.Several approaches that address these biochemical challenges have been developed , and those that generate light such as luciferases are potentially more sensitive for an in vitro assay. Light-generating luciferase enzymes are very sensitive and have been developed for expression and secretion by mammalian cellsHere, we have explored the use of different enzyme reporters for low affinity extracellular protein interaction screening using an ELISA-style direct binding assay with the aim of reducing the amount of protein required, and investigate the possibility of using just a single recombinant protein construct for each protein tested. We report that highly avid constructs based on a flash luciferase—because the luciferase activity was inhibited by exposure to the substrate—enables the enzyme to be used as both bait and prey.28  that produced highly-avid pentamers, and the enzyme beta-lactamase28 Fig. a, b. Wite30 Fig. a. To enae30 Fig. a. To detrey Fig. c; by conrey Fig. c. One po32. We observed that both the Gaussia and nano-luciferases provided a more sensitive detection of the interaction compared to beta-lactamase whilst maintaining a low background signal  which has an equilibrium dissociation constant of 2.5 μM and a dissociation half-life of 0.9 s00-Cd200RGaussia luciferase pentamers on streptavidin-coated microtitre plates and probed them for interactions with a Cd200 beta-lactamase tagged prey. We observed that the interaction could be robustly detected and the sensitivity of the assay increased when compared to immobilised monomer baits  and is not dependent upon a labile or soluble inhibitor that would be removed during wash steps. We observed that once the coelenterazine substrate is added to the Gaussia luciferase enzyme immobilised on the microtitre plate, after an initial very bright burst of light, there is a gradual decline in luminescence that becomes undetectable after an hour   Fig. d. Togeth4. To determine the limit of sensitivity of this assay, we used a set of quantified interactions within a family of six paralogous receptors that belong to the zebrafish junctional cell adhesion molecule (Jam) family that have previously been used to benchmark extracellular interaction screening33. It is known that the interaction between JamB1 and JamC1 is essential for myoblast fusion in vivo34, and the interactions within these receptors have been systematically determined and quantified using surface plasmon resonance. These interactions therefore represent a useful set of both homophilic and heterophilic interactions with a range of affinities from the relatively strong JamB1–JamC2 interaction (t1/2 > 2 s) to the very weak JamA1–JamC2 interaction (t1/2 < 0.4 s), as well as several known negatives35; the expected interactions are depicted graphically in Fig. Gaussia luciferase (non-biotinylated) pentamer prey, and a Gaussia luciferase pentamer biotinylated bait. The proteins were normalised according to the parameters previously established for optimal interaction detection and all 36 possible interactions tested in a pairwise fashion with each Jam bait arrayed in a streptavidin-coated plate, and all six Jam preys probed against them. The original beta-lactamase prey version of the assay performed as previously described35, easily detecting the highest affinity interactions. It also detected all the weaker (t1/2 < 0.4 s) interactions, although two false positives  were identified at this assay stringency . This increase in background may be due to the exchange of biotinylated prey and baits during the prey incubation step or bait protein stripping with the unconjugated biotin wash used to block all remaining biotin binding sites on the streptavidin-coated plate. Although it doesn’t take advantage of using the one-construct system, we did observe that replacing the beta-lactamase enzyme with Gaussia luciferase resulted in very high signal:noise ratios across all the known interactions, including the very weak ones leading to a significant improvement in assay performance. Similarly, while it cannot be inhibited in the same way as the Gaussia luciferase, and can therefore only be used as a prey reporter construct, we found that the “glow” properties of the nano luciferase was a significant improvement on the use of beta-lactamase as a prey reporter enzyme. The increased sensitivity coupled with the long-lasting luminescence made screening a large number of assay plates very practical. Indeed, we have recently used this assay format to investigate the receptor-ligand interactions involved in the recognition of hepatocyte host cells by the liver stage (sporozoite) of the malaria-causing parasite P. falciparum. Using this implementation of the assay, it was possible to produce sufficient protein for a very large receptor interaction screen (88 parasite ligands versus 182 human receptors) by conveniently transfecting cells in 24-well plates rather than larger (250 mL volume) Erlenmeyer flasks39.Both 7 interaction tests. Importantly, this assay should be able to detect weak interactions (KDs ≤ 50 µM) that are a feature of many extracellular interactions4. We found that the use of Gaussia luciferase as a reporter enzyme in a highly avid pentamer construct both reduced the amount protein required and enabled binary interaction screening using a single protein preparation. These refinements have therefore significantly reduced the amount of resources required and will make large-scale extracellular interaction screening less reliant on access to large-scale protein expression infrastructures.Systematically screening for direct interactions using ELISA-style binary binding assays against all ~ 2,000 tractable receptor proteins in a mammalian genome necessitates an assay that has a throughput of ~ 1028. Enzyme and tag sequences were synthesized by gene synthesis (Geneart) essentially as described40. The bait proteins were co-transfected with a plasmid for expression of the E. coli enzyme BirA, which monobiotinylates a specific lysine residue in a C-terminal peptide sequence. The transfected cells were incubated for 5 days (in the presence of D-biotin or hemin where specified at 50 μM). The supernatants were harvested by centrifugation at 3000g for 20 min and either stored at 4 °C until use or purified. Except for HRP constructs, 2 mM sodium azide was used as a preservative for stored supernatants. Where required, proteins were purified from spent tissue culture media using Ni2+-NTA resin using an AKTA pure instrument or HisTrap96 well plates  as described previously41 or according to manufacturer’s instructions. Briefly, plates were spun to remove storage solutions and washed with binding buffer . Supernatants were loaded, spun, and washed three times with binding buffer before being eluted in 500 µl of elution buffer .All proteins were expressed as secreted proteins by transient transfection of the human HEK293E cell line grown in suspension as describedGaussia luciferase, accession number: AAG54095 residues K18 to D185; nano-luciferase, AHH41346 residues V2 to T171; beta-lactamase WP_125075679 residues H19 to W282; horseradish peroxidase P00433 residues Q31 to S338.The following enzyme sequences were used: 28. Briefly, both bait and prey protein preparations were normalised to activities that have been previously shown to detect transient interactions  with a low false positive rate18. Biotinylated baits that had been purified were immobilised in the wells of a streptavidin-coated 96-well microtitre plate (NUNC). Normalised preys were added, incubated for 1 h at room temperature, washed three times in PBS / 0.1% Tween-20, and once in PBS, after which (for the beta-lactamase constructs) 125 µg/mL of nitrocefin was added and absorbance values measured at 485 nm on a Fluostar Optima (BMG laboratories). This method was modified for the alternative prey constructs to suit their enzyme requirements: for Gaussia luciferase, coelenterazine was added at 1 μM (or as indicated); to nano-luciferase, fumerazine was added at 1 μM. Luminescence was measured on a Fluostar Omega (BMG) with no filter and 1 s integration.Binding assays were performed as previously describedobs−Smin) / (Smax−Smin); where ‘S’ is the ‘fraction of maximum signal’ displayed on data plots, ‘Sobs’ is the raw signal in a test well, ‘Smin’ is the average background signal taken from Cd4 negative control bait wells, and ‘Smax’ is the highest signal recorded for the standard interaction at the highest prey concentration. Using this equation, background signal would be 0, and the maximum signal 1.Each of the enzymes compared in this study has a different output whether colorimetric or luminescent with a different signal intensity. To compare them using a normalised signal output that accounted for the background signal, we used the equation S = (S35 were expressed as the monomeric biotinylated bait, the beta-lactamase pentamer prey, the Gaussia luciferase pentamer prey and the Gaussia luciferase pentamer biotinylated bait. Each protein was purified by nickel affinity purification as described41 and normalised according to the parameters established in the standard assay for optimal interaction detection: the prey protein of interest at 100 pM; 1 pmol of mono-biotinylated bait; 10 fmoles of biotinylated luciferase pentameric bait. These constructs were then tested for binding in a pairwise fashion with each Jam bait arrayed in a streptavidin-coated plate, and each set of six Jam preys tested for binding, making a total of 36 binary interaction tests for each prey-bait combination. In the case of the pentamer bait an additional inactivation step using an excess of coelenterazine was used followed by a wash before the addition of prey protein. The beta-lactamase prey was assessed using accumulated hydrolysis of the colorimetric substrate nitrocefin, while the Gaussia luciferase preys were assessed using luminescence shortly after substrate addition.Each of the six Jam proteinsSupplementary information