The setup is optimized for signal handling at the resonance frequencies for 1H (297.2 MHz) and 31P (120.3 MHz). It consists of an eight‐channel 1H transmit–receive head coil with multi‐transmit capabilities, and an insertable, actively detunable 31P birdcage (transmit–receive and transmit only), which can be combined with a seven‐channel receive‐only 31P array. The setup enables anatomical imaging and 31P studies without removal of the coil or the patient. By separating transmit and receive channels and by optimized addition of array signals with whitened singular value decomposition we can obtain a sevenfold increase in SNR of 31P signals in the occipital lobe of the human brain compared with the birdcage alone. These signals can be further enhanced by 30 ± 9% using the nuclear Overhauser effect by B1‐shimmed low‐power irradiation of water protons. Together, these features enable acquisition of 31P MRSI at high spatial resolutions (3.0 cm3 voxel) in the occipital lobe of the human brain in clinically acceptable scan times (~15 min). © 2015 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.The design and construction of a dedicated RF coil setup for human brain imaging ( Moreover, 1H B1 homogeneity becomes a real challenge in larger volumes at higher field strengths (≥7 T), but this is less so for 31P. Rodgers et al found a 2.8‐fold increase for 31P‐MRS in the human heart when going from 3 T to 7 T 31P‐MRS is reduced compared with 1H spectra, because fewer metabolites have a 31P nucleus present, and fewer resonances overlap.The sensitivity of the 31P nuclei. A clever design of this hardware can contribute to an improved signal‐to‐noise ratio (SNR) for detection of these signals. RF surface coils, regardless of their operating frequency, have been frequently used for their excellent, albeit spatially constrained, SNR B1 field, requiring complex high‐power adiabatic pulses to homogeneously excite a region of interest (ROI) close to the coil Dedicated hardware is needed to detect MR signals from 31P RF coil setup needs to be integrated into another RF coil that handles 1H signals. In contrast to clinical MR systems (at B0 ≤ 3 T), no whole body RF coil is provided as standard within human 7 T systems 1H RF coils must be employed to excite the proton spins and receive their signals, to obtain anatomical images and to be able to optimize the field (B0 and B1 shimming). The short wavelengths produce inhomogeneous RF fields; these destructive interferences can coincide within an ROI, decreasing signal intensity at the desired location. When using multiple signal transmission (Tx) coil elements, it is possible to adjust the transmitted RF field by changing input phases and amplitudes of the RF pulses to remove (or shift) these interferences outside the ROI, known as B1 shimming B1 shimming proton signals of water can be irradiated with low RF power, the nuclear Overhauser effect (NOE) 31P nuclei surrounded by water, potentially providing an additional sensitivity to a 31P experiment. This enhancement technique can be used in any organ as long as sufficient power to excite the 1H spins can be delivered to the ROI. Positive NOE enhancements have been reported for prostate and brain 31P MRSI at 7 T 31P compounds, as has been shown for 3 T T2 relaxation during long spin evolution times at 7 T could decrease or even negate the polarization gain.The 31P spin excitation and high SNR 31P MRS(I) for 7 T. This safety‐validated coil setup should allow optimization of the 1H transmit field (B1 shimming) and standard 1H imaging as well as uniform excitation and acquisition of 31P spins throughout the human brain, with a possibility of using an additional local receive array and without removing the coil or the patient during an examination. Therefore, we combined an eight‐channel (8‐CH) multi‐transmit head coil (1H) with a newly constructed and insertable actively detunable volume resonator (31P), and developed a 7‐CH receive‐only (Rx) array (31P) that improves SNR locally. Together with optimized signal addition and the possibility to use B1‐shimmed NOE the sensitivity of local 31P signal detection is maximized.Our aim was to design and construct an integrated coil setup optimized for efficient NOE, uniform 1H transceiver array (TxRx), an actively detunable 31P volume resonator and a 31P 7‐CH Rx array. Note that the 31P volume resonator can be used as transceiver (without Rx array) and as transmit only (with Rx array).The complete coil setup consists of three parts: an 8‐CH 1H head coil designed by Orzada et al. 31P were added to all individual elements.As basis for the complete setup we chose to use an existing 31P coil was designed as a BC coil to allow uniform excitation of the 31P signals in the human brain, which should fit inside the 8‐CH head coil. Therefore, we created an eight‐rung, high‐pass design. This design avoids interference of the resonant modes with the 8‐CH head coil, as the highest useful resonance is the dominant mode 1H microstrip elements . We enlarged the diameter to 25 cm by opening the tube at one side over the complete length. The opening was fixed by two small Plexiglass parts (l × w 3.2 × 3.2 cm2), which supported the end rings. Two rings of copper foil (width 12.5 mm) were attached to the tube with a spacing of 155 mm. Between the end rings, a total of eight rungs  were placed equidistantly around the tube. A smaller width of the rungs compared with the end rings was chosen to minimize distortion of the 1H field  attached to a Plexiglas tube (il width 2.5 mm we31P compounds. Each circular loop (channel) was made from copper wire (wire diameter 1.25 mm2), having a loop radius of approximately 25 mm and having four symmetrically distributed fixed tuning capacitors (68 pF each). Each loop was matched to 50 Ω (loaded) by placing an additional capacitor (162 pF), which was also part of the active detuning network, in the loop. Part of this active detuning is the parallel positioned PIN‐diode. To create a high impedance at the resonance frequency in the loop, a small inductor of 11 nH was added between the PIN‐diode and the matching capacitor , where the amplitude and phase of each channel could be altered using a vector modulator, enabling B1 shimming All experiments were performed on a 7 T whole body MR system . The 8‐CH 31P signal . This signal was divided and phased by a lumped‐element quad‐hybrid to drive the BC in quadrature mode. The 31P receive array was connected to a homebuilt 7‐CH receive interface box optimized for signal acquisition at 120.3 MHz, where in each receive‐path an improved tank circuit A single 8 kW RF amplifier was used to excite the S12) of the BC, by using two loosely coupled pick‐up probes that were connected to a vector network analyzer (VNA) . This setup was also used to determine the ratio of unloaded‐to‐loaded quality factors (QU/QL) The BC was tested in TxRx mode and in Tx mode. In the latter case, the coil must be detuned while receiving the MR signal with the local Rx setup. Active tuning was verified by measuring the transmission coefficient (QU/QL) for each element. Active detuning was checked by switching the currents to the PIN‐diode between forward and reverse bias, while measuring the effect of the S12 response of two pick‐up loops that were weakly coupled to the element under investigation. This method was also used to determine the Q‐factor. Crosstalk between elements was determined by connecting the elements under investigation to the network analyzer, probing signal through one of the elements and receiving with the other. Note that only the element under investigation was tuned and all other coil elements were detuned while making these measurements.For the receive array, we assessed active detuning, determined the crosstalk between each element, and determined the ratio of unloaded‐to‐loaded quality factors . Field probes (Schmid & Partner Engineering AG (SPEAG), Zürich, Switzerland, probes H3DV7 & ES3DV2) were used to collect the maps in a sagittal (AP × FH 190 × 190 mm2) and coronal (LR × FH 160 × 190 mm2) plane in the phantom. Data were collected every 10 mm while probing through the complete coil setup with and without the 31P Rx array insert at the desired frequency (1 W). Both planes were positioned along the midline of the phantom  of the setup were made to assess homogeneity of the 31P signal when acquiring data with the Rx array instead of the BC, (iii) obtaining the noise correlation matrix of the 7‐CH Rx array, and (iv) 1H B1+ shimming with all 31P components present. For these tests a cylindrical phantom (diameter 16 cm and volume ~ 5 L) filled with inorganic phosphate (Pi) (30 mM) (and 2% agar) was used. For an additional in vivo test two healthy volunteers  were scanned after giving written informed consent.The complete functionality of the coil setup was checked with the following tests: (i) 90° flip angle calibration of the BC, (ii) determination of the SNR increase of the TR = 15 s, single shot) selecting a 40 mm thick axial slice through the center of the coil was used for flip angle calibration, where the RF amplitude of the excitation pulse was varied to find the maximum amplitude of the peak present in the phantom (Pi), which approximates a 90° excitation.A repeated slice selective pulse acquire sequence (6 ms excitation pulse (sinc), 31P coils. In both experiments, we excited the 31P signal using the BC, but acquired the signal with the BC in one, and acquired the signals with the 31P Rx array in the other experiment. Signals from the individual receive channels were combined using the WSVD combination method TR/acquisition delay 1000/0.1 ms, flip angle 40°, hard pulse duration 0.3 ms, field of view (FOV) 240 × 240 × 200 mm3, matrix 12 × 12 × 8, elliptical k‐space acquisition with 100% Hamming filter, vector size 1024 and Tacq 5 min 15 s. The spectra from the MRSI data were fitted with Metabolite Report, a work in progress package from Siemens Healthcare . Metabolite Report performs automated, prior‐knowledge‐based, complex fitting in the time domain and has previously been applied to 7 T 31P spectroscopy data of the prostate i signal were created by interpolating its initial 12 × 12 × 8 matrix to a 256 × 256 × 8 matrix. They were then convoluted by a disk‐shaped kernel and masked. These images were then used to determine the local signal gain when using the Rx array as compared with the BC. An additional examination without RF excitation pulses was performed to obtain the noise correlation matrix.Two 3D MRSI data sets of the same cylindrical phantom were obtained to determine the SNR performance of the in vivo study we investigated the ability to use B1 shimming and the ability to acquire 3D 31P MRSI in the human brain with and without NOE enhancement on a single volunteer (24 years) . A high‐resolution 3D 31P MRSI using NOE enhancement with an approximated true voxel size of 3.0 cm3 was acquired from the second volunteer (26 years). This time data was collected with the Rx array, providing a reduced FOV. Parameters used for this sequence were TR/acquisition delay 500/0.10 ms, flip angle 30°, FOV 140 × 140 × 100 mm3, matrix 14 × 14 × 10, NSA = 6 and Tacq 15 min 2 s; all other parameters were kept the same. As the TR is reduced and we want the maximum signal per unit time, we reduced the flip angle to the Ernst angle for PCr (30°) with T1 = 3.4 ± 0.3 s γB1 = 30 Hz) using the wideband alternating‐phase low‐power technique for zero residual splitting  TR, except during the 204 ms of signal acquisition. Other examinations within the protocol were the same for both volunteers. After B0 and B1 shimming the whole brain, a 3D T1‐weighted image was acquired with an MPRAGE (magnetization prepared rapid acquisition gradient echo) pulse sequence. For this sequence the following parameters were used: TR/TI/TE 2500/1100/1.270 ms, resolution 1 mm3, Tacq 3 min 58 s. Then we calibrated the flip angle for 31P, and we optimized the B1 shim for the occipital lobe.In an olunteer  years. TQU/QL) for the BC was 110/30 = 3.7. For the receive array it was (155 ± 20)/(63 ± 5) = 2.7 ± 0.3, with a range of 124–181 for QU and 57–69 for QL.The ratio of unloaded‐to‐loaded quality factors  compared with the |H| field confirmed that it was safe to use this coil assembly with the original 8‐CH safety margins.The proton |ons Fig.  illustra1H coil assembly. For 31P the maximum permissible input power was calculated based on a worst‐case approximation (losses in the feeding network and RF coil were neglected) for the head‐averaged SAR according to the rationale given in the IEC 60601‐2‐33 for volume transmit coils: Pmax31P = SARhead,limitmhead.Comparing these results with the 8‐CH proton setup without any additional coils 31P signals in a phantom as well as in the human brain showed that the maximum signal intensity coincided with a reference B1+ amplitude of 29 μT. This resulted in a maximum achievable B1+ amplitude for 31P of approximately 56 μT for this coil setup .Calibration of the RF power to reach a 90° flip angle for the The noise correlation matrix Fig.  confirmeAs expected, the SNR increased substantially when the Rx array was used for signal acquisition as compared with the BC. An increase in SNR was visible up to 7 cm inside the phantom, with a sevenfold increase up to 2 cm inside the phantom Fig. .in vivo experiments were successful. We could obtain detailed T1‐weighted images with the 8‐CH 1H head coil driven in CP+ mode  in 15 min, with narrow linewidths for PCr (9.6 ± 1.9 Hz) and high SNR (17.8 ± 3.7) , we obtained igh SNR 1.8 ± 3.7 1H and 2.2 W for 31P.The SAR for the experiments with NOE enhancement was evaluated using the real‐time SAR monitoring system 1H head coil, an insertable and actively tunable BC coil for 31P and a local 7‐CH 31P Rx array. The setup featured multi‐transmit capabilities on the 1H channels, volume excitation for 31P, and a large local SNR improvement close to the receive array. This increase in sensitivity in combination with the limited FOV of local receive coils enabled 31P MRSI at higher spatial resolution, and may be used to reduce scan time.In this work, we have described the design, construction, and safety validation of a dedicated coil setup for 7 T containing three main components: an 8‐CH multi‐transmit 1H head coil had to be adapted, because its initial tuning frequency dropped below the resonance frequency of the system. Therefore we tuned it to the correct frequency and adapted the matching to become 50 Ω (loaded) with the BC present. Despite this adaption, we noticed a reduction in sensitivity of the 1H signal, which is probably caused by the large quantity of conductive material present in the BC itself. Although this might block part of the signal, as reported for high‐density receive arrays The tuning and matching of the 8‐CH Although not shown here, it is worth noting that the homogeneity of the BC was hardly altered by the addition of the local Rx array. Each element in the receive array was actively detuned, meaning that a current was used to forward bias a parallel positioned PIN‐diode, bringing the element off resonance during BC transmit. We added no additional safety features, such as a second trap with passive diodes QU (110), and when the QU/QL ratio is above 3.5 tissue loading dominates over coil losses. Hence, the introduction of PIN‐diodes in the BC did not degrade its performance in the first place.The SNR increase by exciting the spins via the BC and receiving their signal with the array coil may have been biased, as the BC for reception may have had degraded performance. The PIN‐diodes for tuning the BC were positioned in series within the legs: when forward biased (tuned) they have a low additional series resistance; when the PIN‐diodes are reverse biased (detuned) this resistance will increase significantly. However, the coil has a relatively high 31P spectroscopy has been reported before 31P MRSI of the patient without removal of the coil or the patient. The advantage of our design over theirs is the ability to use B1 shimming. Moreover, the size of the individual elements of the Rx 31P array in our design was smaller, resulting in a local increase in SNR.Increasing SNR by separating Rx and Tx surface coils for et al. that signal from PCr in the human brain can be increased by 24.3 ± 1.6% using NOE 1H B1 field  are also present, thus the head‐averaged SAR would be reduced even further. Furthermore, when using phased array coils for exciting the MR signal instead of volume resonators, SAR deposition can be reduced significantly (up to 50%) while optimizing the B1+ distribution, as has been shown by van den Bergen et al. in the pelvic area et al. that SAR levels are lower in microstrip coil designs at 7 T 31P, were higher at 7 T. Note that we optimized this BC coil for a lower frequency (120.3 MHz), and when using volume coils at these frequencies the head‐averaged SAR is usually the most critical aspect.Although the amplitude of the 31P in the human brain at 7 T have been using surface coils to excite and receive the MR signal et al. B1 fields are inhomogeneous, hence the flip angle becomes position dependent should one use a hard pulse for excitation. By using adiabatic pulses these limitations can be circumvented, but at a cost of markedly increased pulse duration and concomitant increase in SAR deposition. Using the homogenous volume resonator present here allowed us to avoid SAR demanding adiabatic pulses, but rather use short (0.3 ms) hard pulses that have sufficient bandwidth to excite the spectrum (except β‐ATP). Note that Zhu et al. were not able to use these in their study Most studies on 1H and 31P signals at an ultra‐high field strength of 7 T. 31P signals can be acquired with an increased SNR by exploiting NOE enhancement and by receiving the signals with a local receive array.In conclusion, the developed coil setup can be used to excite


The analysis of gene expression using DNA microarrays provides genome wide profiles of the genes controlled by the presence or absence of a specific transcription factor. However, the question arises of whether a change in the level of transcription of a specific gene is caused by the transcription factor acting directly at the promoter of the gene or through regulation of other transcription factors working at the promoter.a1-α2 complex, which represses haploid-specific genes in the yeast Saccharomyces cerevisiae. Our analysis identified many known or suspected haploid-specific genes that are direct targets of the a1-α2 complex, as well as a number of previously uncharacterized targets. We were also able to identify a number of haploid-specific genes which do not appear to be direct targets of the a1-α2 complex, as well as a1-α2 target sites that do not repress transcription of nearby genes. Our method has a much lower false positive rate when compared to some of the conventional bioinformatic approaches.To address this problem we have devised a computational method that combines microarray expression and site preference data. We have tested this approach by identifying functional targets of the These findings show advantages of combining these two forms of data to investigate the mechanism of co-regulation of specific sets of genes. A bioinformatic approach to identifying cis-regulatory elements controlling transcription has become feasible with the availability of complete genome sequences and large scale expression data using high-throughput methods such as microarrays ,2 and SASaccharomyces cerevisiae 2 - A(Xa(g) - Xα(g))2.Score(g for the three cell types for the variables Xt(g) with t indicating the type. We have since found that using the complete expression data for the polyploids is a better strategy. In the final results, shown in the paper, Xa(g) is the average of the log expression for a, aa and aaa. Likewise, Xα (g) is the average from α, αα and ααα. Xa/α(g) comes from averaging log expression over aaα, aαα and aaαα. The polyploid averaged quantities tend to be less noisy , namely, fraction of genes with score higher than g.An explanation of the purpose served by different terms in the overall score is described below. The first term scores well when expression in diploids is lower than the average expression in haploids. The second term penalizes the gene if the expressions in different types of haploids are very different. a1-α2 repressor complex. In order to select for genes with an upstream region with a strong a1-α2 repressor, we used the binding site mutational data available  ≈ eβE(S)/z assuming near saturation of binding. The symbol z represents the fugacity and β is inverse of kBT, kB being the boltzman constant. The binding (free) energy is given by E(S) = ΣibεibSib, within the single base model )•exp(ΔG(S)/RT) in the biochemistry literature -ATP, and then annealed with excess cold complementary oligonucleotide. The HO(10) and HO(8) a1-α2 sites within Upstream Regulatory Sequence 1 (URS1) of the HO promoter were used as strong and weak binding sites respectively. The EMSA was performed as described previously [μM a1 and five-fold titrations of α2 starting at 82 nM in protein dilution buffer .Oligonucleotides containing the predicted eviously , using aa1-α2 binding sites were synthesized with 5' overhangs to allow cloning into the XhoI site of pTBA23 (2μ URA3 Ampr), a reporter plasmid containing a CYC1-lacZ fusion [β-galactosidase activity was measured on three independent transformants, as described previously [Oligonucleotides containing Z fusion . Reporteeviously .SAGE – Serial Analysis of Gene ExpressionHD – HomeodomainORF – Open Reading FrameChIP – Chromatin immunoprecipitationNHEJ – Non-Homologous End JoiningTC – Total ChromatinIP – Immunoprecipitated sampleEMSA – Electrophoretic Mobility Shift AssayPCR – Polymerase Chain ReactionPMSF – Phenyl Methyl Sulfonyl FluorideEDTA – Ethylenediaminetetraacetic AcidVHN carried out the ChIP experiments, and wrote parts of the first draft of the manuscript. RAO did the bioinformatics analysis, and wrote programs facilitating primer design. ARB did EMSA and beta-gal assays. JRM constructed the strains and contributed to the design of the ChIP experiment. AKV and AMS supervised and coordinated the computational and the experimental research as well as prepared the manuscript. All authors contributed to the manuscript and approved the final version.


The article “A Cross-cultural Perspective on Intrathecal Opioid Therapy Between German and Iranian Patients”, written by Barbara Kleinmann ⋅ Nayereh Khodashenas Firoozabadi ⋅ Tilman Wolter was originally published electronically on the publisher’s internet portal on 28th July 2020 without open access. With the author(s)’ decision to opt for Open Choice the copyright of the article changed on 1st September 2021 to © The Author(s) 2021 and the article is forthwith distributed under a Creative Commons Attribution.The original article has been corrected.


In situ characterizations and labelling experiments unveil water is photoreduced into hydrogen, while non-recyclable plastics including polyethylene bags, polypropylene boxes and polyethylene terephthalate bottles are photodegraded into carbon dioxide, which is further selectively photoreduced into carbon monoxide. In-depth investigation illustrates that the efficiency of syngas production mainly depends on the carbon dioxide reduction process and hence photocatalysts of high carbon dioxide reduction activity should be designed to promote the efficiency of plastic-to-syngas conversion in the future. The concept for the photoreforming of non-recyclable plastics into renewable syngas helps to eradicate ‘white pollution’ and alleviate the energy crisis simultaneously.Plastics take hundreds of years to degrade naturally, while their chemical degradation typically requires high temperature and pressure. Here, we first utilize solar energy to realize the sustainable and efficient plastic-to-syngas conversion with the aid of water at ambient conditions. As an example, the commercial plastic bags could be efficiently photoconverted into renewable syngas by Co–Ga Non-recyclable commercial plastics are converted into syngas with solar as energy source in pure water at ambient conditions, which helps to alleviate ‘white pollution’ and the energy crisis simultaneously. The atomic force microscopy image in Fig. S3B and C indicates their average thickness of ∼1.05 nm, consisting well with the thickness of 12 atoms along the [1 –1 0] direction of γ-Ga2O3. More importantly, the annular dark-field TEM image and element mapping in Fig. S4 depict the homogeneous distribution of Ga, O and Co elements, while their X-ray photoelectron spectra of the Co 2p core level in Fig. S5 exhibits two feature satellite peaks at 787.1 and 804.1 eV, verifying the introduction of Co2+ in the synthetic sample [2O3 nanosheets with the same orientation and thickness were also fabricated by virtue of a similar synthetic strategy . Furthermore, UV–vis diffuse reflectance spectra and synchrotron-radiation photoemission spectroscopy were utilized to unravel the electronic band structures for the Co–Ga2O3 nanosheets and the Ga2O3 nanosheets . Based on these results, it was demonstrated that the VB edge and the CB edge for the Co–Ga2O3 nanosheets were located at 2.50 and –1.40 V vs. NHE at pH = 7, while the VB and CB edges for the Ga2O3 nanosheets were located at 3.19 and –1.46 V vs. NHE at pH = 7. Thus, one can conclude that both of them are capable of realizing some key reactions such as H2O oxidation or CO2, O2 and H2O reduction [To this end, Co–Ga113) and 2 planes eduction ,24.2) at ambient temperature and pressure. Commercial plastic products of PE plastic bags, PP plastic boxes and PET plastic bottles were initially shredded into powders with a size of <5 mm using a pulverizer . It should be mentioned that small plastics (≤5 mm) were commonly defined as the microplastics, which were particularly difficult to be recycled and may cause some unpredictable damage to the ecosystem. As displayed in Fig. 2O3 nanosheets and the Ga2O3 nanosheets, in which the gas products of H2, CO and CO2 were detected by gas chromatography, while there was no detectable liquid product confirmed by the 1H NMR spectra in Fig. S13. Interestingly, there was plenty of CO2 dissolved in the water (Tables S5 and S6). It is worth noting that a handful of microplastics with smaller size were also detected , which might have been produced during the photoconversion processes, conforming to the principle of carbon balance (please see the Methods section). Taking the commercial PE plastic bags as an example, the evolution rates of H2, CO and CO2 for the Co–Ga2O3 nanosheets were 647.8, 158.3 and 419.3 μmol g–1 h–1—roughly 1.6, 1.9 and 1.6 times higher than those of the Ga2O3 nanosheets, implying the former's better photoconversion performance. Note that the weight loss for the commercial PE plastic bags was 53% after 24 h irradiation over the Co–Ga2O3 nanosheets and the weight loss could reach 81% after 48 h irradiation . More importantly, upon adding another 100 mg of commercial PE plastic bags to the photocatalytic system after 24 h irradiation, the Co–Ga2O3 nanosheets also possessed almost the same formation rates of H2, CO and CO2 , suggesting their superb photocatalytic stability. Consequently, the commercial plastic products including PE plastic bags, PP plastic boxes and PET plastic bottles could be efficiently photodegraded into CO, while H2O could be photoreduced into H2 by the Co–Ga2O3 nanosheets.To disclose whether these two photocatalysts can realize the sustainable conversion of non-recyclable plastics into clean renewable syngas under mild conditions, the commonly used commercial plastic products of PE plastic bags, PP plastic boxes and PET plastic bottles were taken as the examples to perform the photocatalytic experiments, wherein the synthetic samples and the commercial plastics were mixed in pure water and irradiated by simulated sunlight —slightly higher than those for the commercial PE plastic bags. Of note, control experiments showed that H2 was detected, while there was no detectable CO and CO2 without reagent-grade PE in the photocatalytic system, suggesting that H2 may have come from the reduction of H2O (Table S3). To further disclose the origin of the H2, the photoreforming experiment of reagent-grade PE over the Co–Ga2O3 nanosheets was performed in the pure D2O solvent. Note that the synchrotron-radiation vacuum UV photoionization mass spectrometry (SVUV-PIMS) in Fig. 2 was detected in the D2O solvent, which firmly affirmed that the generated H2 originated from the H2O rather than the PE during the photoreforming processes. Moreover, when the light or photocatalyst or reagent-grade PE was removed, there was no detectable CO and CO2 (Table S3), verifying that CO and CO2 originated from the photodegradation of PE by the photocatalyst. Furthermore, H2 and O2 could be detected when no PE powders were added into the photocatalytic system under a N2 atmosphere , implying that the H2O splitting could have been triggered by these two photocatalysts, in which some amount of H2O2 was also produced during the process . To further testify the origin of the CO2 and CO, AgNO3 was added into the solution, which helped to clearly identify the photo-oxidation products since AgNO3 was usually considered as a trapping agent to efficiently consume the photo-generated electrons [2 was detected , further confirming that H2O and O2 participated in the oxidation of PE into CO2. From the above-mentioned results, we deduce that the H2 stemmed from the H2O instead of the PE, while both the O2 and H2O took part in the oxidation of PE into CO2 and then the formed CO2 could be further reduced to CO.To unveil the origin of the generated products, the photoreforming of reagent-grade PE was carried out over the Co–Galectrons ; during in situ electron spin resonance (ESR) spectra were employed to identify the reaction intermediates, where 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used as the spin-trapping agent. The ESR signals in Fig. S22 exhibit a 1 : 2 : 2 : 1 quartet pattern in the water solution, which could be assigned to the ·OH captured by the DMPO [218O isotope-labelling experiments in Fig. 2O would be oxidized into ·OH radicals by the photo-induced holes and then the ·OH radicals would participate in the oxidation processes of the PE. Meanwhile, the ESR signals in Fig. S23 show a quartet pattern with an intensity of nearly 1 : 1 : 1 : 1 in the methanol solution, which could be assigned to the superoxide radicals (O2•−) captured by the DMPO [2O2 [2 is in favor of the photodegradation of PE powders, it is possible that the O2 may undergo stepwise photoreduction into O2•–, H2O2 and H2O. Besides, the detection of C16O18O in both the H218O and 18O2 labelling experiments suggested that both the ·OH radicals and the O2 participate in the photo-oxidation of PE into CO2. To gain in-depth investigation of the CO formation process, photocatalytic experiments in pure CO2 were conducted over the Co–Ga2O3 nanosheets, in which in situ FTIR spectra were performed to trace the reaction intermediates. As revealed in Fig. –1 under irradiation could be assigned to the *COOH and *CO groups, respectively [2 molecules were initially reduced into COOH* intermediates by the incoming proton–electron pair (H+ + e–); then, the COOH* intermediates might have coupled with another H+ + e– pair to form the *CO intermediates, which would be liberated from the surface of the catalyst to form free CO molecules [To further understand the PE photoreforming processes, the DMPO . Combinethe DMPO . As reveMPO [2O2 . Consideectively ,30. It molecules .2O into syngas could be proposed as the following procedure. Under light irradiation, the photocatalysts initially generated electrons and holes pairs, which could react with the H2O to form H2 and O2. Then, the ·OH radicals derived from H2O and the produced O2 or O2 in the air atmosphere could synchronously photodegrade plastics into CO2; and meanwhile, O2 was also reduced to O2•–, H2O2 and H2O in order by the photoexcited electrons. Subsequently, the generated CO2 would be further reduced into CO through *COOH intermediates, while H2O was oxidized into O2. In short, photoconverting commercial plastics and H2O into syngas may undergo the following three steps in sequence , which indicated that the former could make better use of the photon energy and generate more charge carriers to engage in the plastics photoreforming processes [2O3 nanosheets possessed distinctly enhanced density of states at the VB edge , which contributed to the promoted photocarrier transfer ability, and hence helped to provide more photo-generated electrons and holes to participate in the subsequent redox reactions [2O3 nanosheets, indicating the restrained recombination efficiency of photo-generated electron–hole pairs  [2 temperature-programmed desorption spectra in Fig. S27 manifested that the onset desorption temperature of CO2 for the Co–Ga2O3 nanosheets (88°C) was higher than that of the Ga2O3 nanosheets (76°C), suggesting that the former has stronger adsorption behavior for CO2, which is beneficial to the further reduction of CO2 [2 photoreduction into CO was the formation of *COOH intermediates, wherein the Co–Ga2O3 nanosheets possessed a lower COOH* intermediates formation energy compared with the Ga2O3 nanosheets. This could be attributed to the fact that the introduction of Co atoms caused the charge accumulation of the Co atoms and the neighboring Ga atoms, which was conducive to stabilizing the *COOH intermediates through Co–Ga dual active sites and hence lowered its formation energy  . Importan of CO2 . Further2O is photoreduced into H2, while non-recyclable plastics including PE plastic bags, PP plastic boxes and PET plastic bottles are photodegraded into CO2, which is further selectively photoreduced into CO. As an example, commercial PE plastic bags could be efficiently photoconverted into syngas with the aid of H2O by the Co–Ga2O3 nanosheets, in which the H2 and CO formation rates were ∼647.8 and ∼158.3 μmol g−1 h−1 —roughly 1.6 and 1.9 times higher than those of the Ga2O3 nanosheets, respectively. More importantly, the weight losses of PE plastic bags, PP plastic boxes and PET plastic bottles were up to approximately 81%, 78% and 72% after 48 h irradiation over the Co–Ga2O3 nanosheets. Through deeply exploring the mechanism of plastics photoconversion into syngas, it can be concluded that the whole efficiency is mainly dependent on the process of CO2 reduction into CO, and hence it is necessary to design photocatalysts of high CO2 reduction activity to promote the efficiency of non-recyclable plastics degradation in the future. Meanwhile, considering that the residual microplastics in aqueous solution are hard to fully degrade, some precautionary measures such as filtration units and advanced wastewater treatment technologies like membrane filtration may be needed in the future. Briefly, the design concept may help to open up new avenues toward the curbing of white pollution and the relieving of the energy crisis simultaneously.In conclusion, we first realized the sustainable and efficient conversion of non-recyclable plastics into renewable syngas in pure water at ambient temperature and pressure. In this process, H3)3 and 29 mg Co(NO3)2 · 6H2O were added to 5 mL water with vigorous stirring for 10 min. Then, 30 mL triethylenetetramine (TETA) was added to the solution after continuous magnetic stirring for another 30 min. Finally, the solution was transferred to a stainless Teflon-lined autoclave, which was sealed and maintained at 160°C for 48 h. The product was collected by centrifugation, washed thoroughly with absolute ethanol and deionized water several times, and then dried at 60°C for 12 h.In a typical synthetic procedure, 256 mg Ga2 · 6H2O was added.The procedures were the same as those for the Co–Ganwac011_Supplemental_FileClick here for additional data file.


Transforming growth factor-β (TGFβ) is a pleiotropic cytokine that is central in the regulation of joint health and disease. Inhibition of TGFβ activity/signaling in experimental osteoarthritis (OA) has been performed to modulate OA severity and progression. In this narrative review we discuss the potential reasons for the variable results of TGFβ inhibition in these models.A literature study was performed using the search terms; experimental osteoarthritis and TGFβ. Papers were selected that describe the effect TGFβ activity/signaling inhibition on experimental OA. Based on the selected papers a narrative review has been written about the potential therapeutic role of TGFβ inhibition in OA and potential causes for its variable effects are discussed.Inhibition of TGFβ activity in experimental models of OA does not result in either straightforward protection or deleterious effects. More than half of the studies (13/19), but not all, report that inhibition of TGFβ in experimental OA reduces OA severity. This is in contrast with the protective role of TGFβ in healthy joints.The effect of TGFβ inhibition on joint damage in experimental OA is variable. Most likely this is a consequence of the changing function of TGFβ in normal and OA joints. As a result, the overall outcome of TGFβ modulation in OA will be unpredictable. To develop OA therapies based on modulation of TGFβ activity specific protective and damaging signaling routes should be identified and tools developed to block the damaging ones. The etiTransforming growth factor-β (TGFβ) is a pleiotropic cytokine that is important in the regulation of joint homeostasis and disease. In articular cartilage, TGFβ signalling is induced by loading and has an important function in maintaining the differentiated phenotype of articular chondrocytes and dampening of inflammation . SynoviaFunctional TGFβ is a 25 kD dimer that signals via heteromeric complexes of transmembrane serine/threonine type I and type II receptors, most frequently tetramers composed of two type I and two type II receptors .The canonical signalling route of TGFβ is via the broadly expressed type I receptor activin-like kinase 5 (ALK5), but in chondrocytes and other cell types additional ALKs, such as ALK1 , can be involved . Upon tyIn addition to the above described canonical (SMAD-dependent) signaling pathways alternative pathways (SMAD-independent) are described. Recently, a review on non-canonical pathways in TGFβ has been published . TGFβ haIn articular cartilage, TGFβ is stored in latent form in large amounts (∼300 ng/ml) and compressive loading rapidly activates TGFβ signalling in cartilage explants, implying that under normal physiological conditions (regular loading) articular cartilage has a basic level of TGFβ signalling and expression of TGFβ-regulated genes ,14. MutaAlthough loss of TGFβ signalling is deleterious for healthy articular cartilage, in OA joints another situation can occur. Synovial fluid and tissues of OA joints will contain elevated levels of active TGFβ compared to healthy joints. Elevated TGFβ levels most likely contribute in OA joints to synovial fibrosis, osteophyte formation, subchondral bone changes and detrimental changes in chondrocyte phenotype and function. Considering the context dependent function of TGFβ in normal and OA joints the outcome of either inhibition or supplementation of TGFβ in experimental OA is hard to predict. In this article an overview will be given on the effect of TGFβ signaling modulation in experimental OA models and the consequences for the use of TGFβ-dependent OA therapy will be debated.2Inhibition of TGFβ activity during OA in animal models results in many instances in inhibition of OA severity and progression . One of Tgfbr2 in mature condylar cartilage significantly delayed the progression of the cartilage degeneration induced by a partial discectomy. Increase in the expression and activity of TGFβ signaling has detrimental effect on mature condylar cartilages .,55.54,55Besides changes in canonical signaling also disease dependent alterations in non-canonical can play a role. Altered non-canonical signaling can change the function of TGFβ in osteoarthritis. An important factor might be the presence and the magnitude of inflammation in the joint. Inflammation has the potential to modulate the role of TGFβ in the joint by its own effects or by altering SMAD signaling by modulation of the SMAD linker domain .The differential action of TGFβ in a healthy and an OA joint poses a problem for anti-TGFβ treatment. Systemic inhibition will have differential effects on different joints, depending on its disease status and local TGFβ concentrations. As a result, local modulation of TGFβ activity should be the preferred action to treat OA joints. Though, even within a joint TGFβ might have other effects on relatively normal cartilage and severely diseases tissue depending on the local active TGFβ levels.6A factor that also might affect the outcome of treatment is effectivity of anti-TGFβ inhibition. The levels of active TGFβ that remain in the joint will determine what will be the final outcome. Partial inhibition of elevated TGFβ levels can be expected to protect the joint while effective blocking, reducing the active levels to (nearly) zero, could be damaging to the joint. Furthermore, the active concentrations will not be constant but fluctuate depending on the momentary inhibition of active TGFβ.Another aspect that might modulate the outcome of anti-TGFβ treatment on a theoretical base is the timing of the administration of anti-TGFβ. Is a therapeutic or preventive regime used? In a preventive regime, TGFβ will be blocked from the moment of OA induction while a therapeutic regime will start when structural OA changes are developing. In the study of Scharstuhl, finding a deleterious effect of TGFβ inhibition, blocking TGFβ activity was started before OA induction . BSHX fo7Different models of experimental OA are used to test the effect of TGFβ inhibition on joint pathology. All reported studies are in small animal models  in stifle joint (knee) or TMJ, and perform chemically-induced or surgically-induced OA or models based on genetic modification or spontaneous mutations. All these models represent a different aspect of human OA and differ in their translatability to human disease. None of the model covers the whole spectrum of human OA. Nonetheless the variability of these models, most studies report a positive effect of TGFβ inhibition on OA severity. This indicates that in the majority of investigations TGFβ has a similar role in the OA disease process in these experimental models.Responses to TGFβ are age-related and studies in old mice and cows have revealed an age-related decrease in ALK5 expression and a subsequent increase in the ALK1:ALK5 ratio . RecentlThe outcome of the studies will also depend on the target tissue and the OA scoring system used. Inhibition of TGFβ can have different effects on different joint tissues, for instance articular cartilage or subchondral bone. It has been shown that robust inhibition of TGFβ activity (SB-505124) improved subchondral bone structure but induced proteoglycan loss in articular cartilage . Local T8The most striking observation is that inhibition of TGFβ activity/signaling in a normal joint results in joint damage while in most studies, but not all, inhibition of TGFβ in experimental models of OA reduces OA severity. Development of a therapy based on the observed reduction of OA severity by blocking TGFβ activity could be attractive but is complicated by the effect that TGFβ will have on normal cartilage in non-OA affected joints or even within the OA affected joint itself. This makes simple inhibition of TGFβ activity not the preferred option. A better alternative is the development of therapeutic tools that can separate the different TGFβ signaling pathways, blocking the deleterious pathways and concomitantly preserving or stimulating the protective pathways. This means blocking specific receptor complexes or intracellular pathways, a challenging but not impossible option.I have no competing interests with regard to his manuscript. The manuscript is designed and drafted by the first and sole author.


The RAD9–RAD1–HUS1 (9–1–1) clamp forms one half of the DNA damage checkpoint system that signals the presence of substantial regions of single-stranded DNA arising from replication fork collapse or resection of DNA double strand breaks. Loaded at the 5′-recessed end of a dsDNA–ssDNA junction by the RAD17–RFC clamp loader complex, the phosphorylated C-terminal tail of the RAD9 subunit of 9–1–1 engages with the mediator scaffold TOPBP1 which in turn activates the ATR kinase, localised through the interaction of its constitutive partner ATRIP with RPA-coated ssDNA. Using cryogenic electron microscopy (cryoEM) we have determined the structure of a complex of the human RAD17–RFC clamp loader bound to human 9–1–1, engaged with a dsDNA–ssDNA junction. The structure answers the key questions of how RAD17 confers specificity for 9–1–1 over PCNA, and how the clamp loader specifically recognises the recessed 5′ DNA end and fixes the orientation of 9–1–1 on the ssDNA. Substantial segments of single stranded DNA generated by stalled replication forks or resection of DNA double-strand breaks, trigger a DNA damage checkpoint response, which is dependent on activation of the PIKK-family protein kinase ATR and phosphorylation of its downstream target CHK1  mediatedFull catalytic activation of ATR at sites of ssDNA is dependent on the RAD9–RAD1–HUS1 (9–1–1) heterotrimeric checkpoint clamp  preferenThe presence of the 9–1–1 clamp loaded at a 5′-recessed dsDNA–ssDNA junction, is communicated to ATR localised on RPA-bound ssDNA, by the scaffold protein TOPBP1 , whose BThe first structure of the RFC clamp loader, as a complex with PCNA was determined over a decade ago using X-ray crystallography and proteins from budding yeast . More reSaccharomyces cerevisiae were published  clamp loader complex bound to the closed 9–1–1 checkpoint clamp and engaged with a dsDNA–ssDNA junction. The structure explains how replacement of RFC1 with RAD17 changes the loading specificity of the RFC complex from PCNA to 9–1–1 and reveals the means by which the orientation of 9–1–1 loading at a dsDNA–ssDNA junction is dictated through recognition of the 5′ recessed end by RAD17. During revision of this manuscript, two separate papers describing structural studies of the homologous Rad24–RFC–Rad17–Mec3–Ddc1 complex from ublished ,33. ThesSf9 cells with a baculovirus coding for a construct containing RAD9–HIS, RAD1–STREP–RAD17–HIS HUS1 RFC2 RFC3 RFC4 HIS–RFC5 produced using the biGBac system  of the resulting micrographs calculated using MotionCor2  were glow discharged using a Tergeo Plasma Cleaner (Pie Scientific) with an indirect plasma treatment for 30 seconds. Grids were loaded into a Leica EM GP2 (Leica microsystems) and 3 μl of a 600 nM sample was applied to the front of the grid, with an additional 0.5 μl buffer applied to the grids rear, before back blotting for 4 s and plunging into an ethane propane mix. Grids were stored in liquid nitrogen prior to imaging at 300 kV on a FEI Titan Krios (Thermo Fisher Scientific) equipped with Falcon 4 detector (Thermo Fisher Scientific). 8271 movies were collected, using data acquisition software EPU (Thermo Fisher Scientific), at a magnification of 96000× and a pixel size of 0.86 Å using a total dose of 39.4 etionCor2  and CTFFtionCor2  implementionCor2 . All subThese were refined in 3D and sharpened to give a model with a resolution of 5.16 Å estimated by ‘gold standard’ Fourier Shell Correlation . RefinedSf9 insect cells, then purified by affinity and size exclusion chromatography . The yields of assembled complex in initial trials were low due to the apparent lability of the human RAD17 protein, which was readily lost from the otherwise very stable RFC2–RFC3–RFC4–RFC5 clamp loader core. To circumvent this, we explored a fusion strategy in which RAD17 was genetically fused via an extended ‘linker’ to the N- or C-terminus of either RAD9, RAD1 or HUS1. While some fusions completely disrupted assembly, fusion of RAD17 to the C-terminus of RAD1 significantly stabilised the overall complex, and delivered material in which all subunits co-purified, which co-migrated with a dsDNA–ssDNA junction in electrophoretic mobility assays, and that showed the presence of both clamp and clamp loader in negative stained electron micrographs (data not shown).To obtain protein for structural analysis human 9–1–1 checkpoint clamp and RAD17–RFC clamp loader were expressed together in For cryoEM, the purified samples were mixed with ATPγS and a DNA molecule that folds back on itself to give 24 base-pairs of fully complementary dsDNA capped with a tetraloop at one end, and with a 24 nucleotide 3′ overhang at the other . Samples were applied to grids and images collected on an FEI Titan Krios microscope equipped with a Falcon IV detector in counting mode . Movies were motion corrected and images processed in cryoSPARC  and RELIPreliminary inspection of the post-processed map confirmed the presence of both the 9–1–1 clamp and the RAD17–RFC clamp loader, as well a segment of dsDNA protruding from the core of the particle. As the visible duplex DNA and some clear α-helices had a left-handed thread, the map was inverted to the correct hand Figure .An initial model for the 9–1–1 ring, which appeared fully closed, was obtained by placing the coordinates of our previously determined crystal structure of 9–1–1 (PDB code: 3G65)  into the but making no contact with HUS1 Figure . RAD17–RS)-phosphate group being present, indicating that the state of the clamp-loader observed here corresponds to that prior to the hydrolysis of ATP. The presence of this moiety in the RFC3 subunit (Rfc5 in yeast) agrees with one of the deposited yeast Rad24-RFC complex structures, where additional density ascribed to a thiophosphate is also present . During the review process (for this manuscript), both papers were subsequently accepted for publication and coordinates released, allowing a comparison of the human and yeast complexes.While this work was in progress, pre-prints describing structural studies of the homologous Rad24–RFC–Rad17–Mec3–Ddc1 complex from the yeast Micrographs, CryoEM maps and refined coordinates have been deposited in EMPIAR, the EMDB and Protein Databank as EMPIAR-11062, EMD-14527 and PDB ID 7Z6H respectively.gkac588_Supplemental_FileClick here for additional data file.


The camelid single domain antibody, referred to VHH or Nanobody, is considered a versatile tool for various biotechnological and clinical applications because of its favorable biophysical properties. To take advantage of these characteristics and for its application in biotechnology and therapy, research on VHH engineering is currently vigorously conducted. To humanize a camelid VHH, we performed complementarity determining region (CDR) grafting using a humanized VHH currently in clinical trials, and investigated the effects of these changes on the biophysical properties of the resulting VHH. The chimeric VHH exhibited a significant decrease in affinity and thermal stability and a large conformational change in the CDR3. To elucidate the molecular basis for these changes, we performed mutational analyses on the framework regions revealing the contribution of individual residues within the framework region. It is demonstrated that the mutations resulted in the loss of affinity and lower thermal stability, revealing the significance of bulky residues in the vicinity of the CDR3, and the importance of intramolecular interactions between the CDR3 and the framework‐2 region. Subsequently, we performed back‐mutational analyses on the chimeric VHH. Back‐mutations resulted in an increase of the thermal stability and affinity. These data suggested that back‐mutations restored the intramolecular interactions, and proper positioning and/or dynamics of the CDR3, resulting in the gain of thermal stability and affinity. These observations revealed the molecular contribution of the framework region on VHHs and further designability of the framework region of VHHs without modifying the CDRs. 7XL0 and 7XL1;PDB Code(s):  In a previous study, Cecil Vinkle et al. proposed two general strategies to humanize a VHH: CDR‐Grafting and resurfacing (veneering).The VHH framework segments exhibit high sequence homology to human VH sequences.In this study, we investigated the impact of CDR‐Grafting using a humanized VHH of therapeutic interest on the affinity for the antigen, its thermal stability, and its structures. Herein we revealed the molecular mechanisms explaining the functional loss of the chimeric VHH upon CDR‐grafting. Further, we analyzed the effect of mutations in framework residues, showing the importance of intramolecular interactions between the CDR3 and framework‐2. In light of these observations, we discuss the contribution of framework on the functionality of VHHs from a molecular perspective, and suggest pathways to engineer humanized VHHs with desired properties.22.1Lama glama.We selected a humanized VHH, Vobarilizumab, as a model VHH containing a humanized VHH framework. Vobarilizumab, binding interleukin‐6 receptor (IL‐6R), was developed by Ablynx for the treatment of autoimmune diseases and confirmed its low immunogenicity through clinical trials.Tm) of 7D12‐Vob (52.2°C) compared with both, wild‐type 7D12 (63.6°C) and Vobarilizumab (70.9°C)  Figure , Table 12.2To gain insight into the molecular basis by which CDR‐Grafting diminished functional and physicochemical properties of the chimeric VHH, we determined the crystal structures of 7D12‐Vob and Vobarilizumab in the unbound state. Although the framework region of 7D12‐Vob exhibited a similar structure to that of wild‐type 7D12 (RMSD Cα: 0.63 Å), the CDR3 loop of 7D12‐Vob adopted an extended conformation different from that of wild‐type 7D12,2, 26.1 Å2, and 53.0 Å2, respectively). On the other hand, only Tyr42 among these three positions  was involved in contacts with the CDR3 in the crystal structures of 7D12‐Vob and Vobarilizumab, and with lower values .We calculated buried surface area (BSA) using PDBePISAWe also collected the circular dichroism (CD) spectra of the VHHs to assess the effects of CDR‐Grafting on the structure of VHH in solution. The results showed that the CD spectrum of 7D12‐Vob exhibited a different profile from that of wild‐type 7D12 or from that of Vobarilizumab Figure . This di2.32 as calculated by PDBePISA). We hypothesized that the substitution of a residue with a small sidechain with another residue displaying a bulky sidechain could affect the conformation or dynamics of the CDR loop. To evaluate the effect of the substitution of this position, we prepared the mutant A106F, and analyzed the physicochemical properties of the mutant in the wild‐type 7D12 antibody. The results showed that the A106F substitution resulted in a loss of affinity (~fivefold) and lower thermal stability  located at the end of framework‐3 and at the base of CDR3 as a possible factor influencing the conformation of CDR3. In wild‐type 7D12 the identity of the residue at position 106 is Ala, whereas in Vobarilizumab it is Phe, a residue with a bulky and hydrophobic sidechain. The crystal structure of wild‐type 7D12 in complex with the cognate antigen EGFR2.4We sought to further elucidate the factors affecting the conformation of CDR3, focusing on a structural feature in the VHH long CDR3. As mentioned above, the long CDR3 in VHHs often bends, making contacts with residues of framework‐2, and shielding the hydrophobic surface that corresponds to the interaction region of the VL domain in conventional VH‐VL antibodies.To quantitatively evaluate the contribution of the contact region between CDR3 and framework‐2 at the atomic level, we performed molecular dynamics (MD) simulations using the structure of wild‐type 7D12. The interaction energies, namely Lennard‐Jones potential and Coulomb potential, between CDR3 and each residue on framework‐2, were calculated using the gmx_energy module. Large interaction energies were observed for residues 42 and 52, among residues at the framework‐2  and also the double mutant (F42Y‐F52L), and carried out the same biophysical analysis outlined above . Introduction of a single mutation in wild‐type 7D12 led to a two‐fold affinity loss with the antigen Figure , Table 22.6and Leu52, and Phe106 of 7D12‐Vob were mutated to Phe42, Phe52, and Ala106, respectively.As observed above in the antibody 7D12, CDR grafting often results in partial or complete loss of affinity of chimeric antibodies. In such cases, back‐mutations, in which residues of the chimeric antibody are replaced with the corresponding residues in the parental antibody, have been attempted to restore the affinity and stability lost.KD: 191 and 110 nM, respectively) than L52F mutant (KD: 358 nM) suggested larger effects of the mutation at position 42 on affinity , suggesting that residues at position 42 and 52 cooperatively stabilize the chimeric VHH  and thermal stability (3–5°C) Table . As we iKD: 4.7 and 4.1 nM respectively), thus recovering the affinity lost upon CDR‐Grafting.Although partial recovery of affinity was observed in the individual mutants, the triple mutant 7D12‐Vob Y42F‐L52F‐F106A performed below expectations, exhibiting a 10‐fold lower affinity than the wild‐type 7D12. As additional potential factors explaining the poorer performance, we found that N‐terminal Gln formed a hydrogen bond with the EGFR extracellular domains from the co‐crystal structure of the wild‐type 7D12 and EGFR extracellular domains.3In this study, we assessed the effects of humanization of VHH via CDR‐Grafting. While various humanization campaigns on VHHs have been reported using CDR‐Grafting,We investigated the structural aspects of chimeric VHH using crystal structures, MD simulations, and CD spectra to elucidate the molecular basis explaining the suboptimal functionality of the VHH upon CDR‐grafting. Although small conformational changes of CDR loops in antibody humanization have been observed in previous research,The mutation of a residue located at the base of CDR3 (A106F mutation) also resulted in loss of affinity and thermal stability. This mutation indirectly contributes to the interaction, probably by hampering the proper positioning and/or dynamics of the CDR3. As shown in previous studies,Our analysis using MD simulations quantitatively described the contribution of residues at positions 42 and 52 to the intramolecular interactions between CDR3 and framework‐2. Introducing mutations into these positions  resulted in significant loss of affinity and thermal stability, especially in the double mutant (7D12 F42Y‐F52L), suggesting that these two residues, Phe42 and Phe52, cooperatively contributed to its own stability  and affinity for the antigen. In addition, the CD spectra showed significant effects on the secondary structure of the VHH when mutating residue Phe42 (F42Y mutation). These results suggested the distinct roles of Phe42 and Phe52: Phe42 would play a critical role to maintain the bent conformation of the CDR3, whereas Phe52 would support and stabilize the role of Phe42. While Phe42 would display van der Waals contacts with the atoms in CDR3, our results implied that the mutation F42Y, adding of a hydroxyl group, could disturb these van der Waals contacts.In the case of VHHs with long CDR3 folding toward the framework, disulfide bond formation between CDR3 and framework‐2, or a hydrogen bond formation between the CDR3 residue and Arg50 at framework‐2 was also observed.In addition to the intramolecular interactions, we also described two additional factors resulting in poorer performance of 7D12‐Vob compared to the parental VHH. As we discussed above, the back‐mutation at the base of CDR3 (F106A mutation) increased the affinity and thermal stability, suggesting that the mutation modulated the proper positioning and/or dynamics of the CDR3. Moreover, an increase in affinity upon introducing a back‐mutation at N‐terminus (E1Q mutation) indicated that interaction between the VHH and EGFR was mediated not only by CDRs but also by the N‐terminal residue.Finally, we demonstrated that the affinity and thermal stability of the VHH could be improved by introducing mutations in the framework region without modifying the CDRs. These results provide important insights into the optimization strategy of humanized framework of VHHs, and moreover as a general strategy for VHH engineering in which the sequence of CDRs should be preserved to maintain their affinity and specificity. Further studies will expand the designability of VHHs based on our exhaustive study.44.1Escherichia coli BL21(DE3) cells  were transformed with the plasmid constructs. The recombinant VHHs were extracted from a periplasmic fraction of E. coli using ultrasonication, and purified using Ni‐NTA agarose  equilibrated with the binding buffer . The VHHs were eluted with the binding buffer containing increasing concentrations of imidazole (20–500 mM). The eluted fractions were further purified by size‐exclusion chromatography using a HiLoad 26/600 Superdex 75 pg column  equilibrated with PBS buffer.The DNA sequence encoding each of the VHHs with a C‐terminal hexa‐histidine tag was cloned into an expression vector, pRA2, for bacterial expression. To generate 7D12‐Vob or introduce mutations, a standard inverse PCR method was performed using primers containing each CDR sequence or each of the mutations. The expression and purification of the recombinant VHHs ware carried out based on a previous report.The DNA sequence encoding the extracellular domain of EGFR (residues 1–618) with an octa‐histidine tag at the C terminus was subcloned into the pFASTBac1 vector . The extracellular domain of EGFR was expressed using Sf9 expression system .The protein concentration was determined by absorbance at 280 nm using the computed extinction coefficient of each protein from their amino acid sequence.4.2Purified Vobarilizumab was concentrated using Amicon Ultra MWCO 10,000 (Merck) to 13.4 mg/ml in PBS. The crystallization was performed in the hanging‐drop vapor‐diffusion method at 20°C. Crystals of Vobarilizumab were obtained by mixing drops of 2 μl of protein with 2 μl of the crystallization buffer and equilibrating against the crystallization buffer composed of 0.2 M lithium sulfate and 20% PEG 3350. Separately, purified 7D12‐Vob was concentrated to 6.5 mg/ml in PBS. The crystallization screening was performed by using an Oryx8 protein crystallization robot  with PEG/Ion and PEG/Ion 2 screening kits . Protein sample (0.5 μl) was mixed with each crystallization buffer (0.5 μl) and equilibrated against a reservoir of the same buffer in the sitting‐drop vapor‐diffusion method at 20°C. Crystals of 7D12‐Vob appeared in a solution containing 0.2 M sodium malonate (pH 4.0) and 20% PEG 3350. Suitable crystals were incubated in a solution containing mother liquor supplemented with 20% glycerol, and subsequently transferred to liquid nitrogen for storage until data collection.Diffraction data from single crystals were collected in beamline BL1A at the Photon Factory in Tsukuba (Japan) under cryogenic conditions (100 K). Diffraction images were initially processed with MOSFLM,4.3The interactions of the VHHs with EGFR were analyzed by SPR using a Biacore 8 K instrument (Cytiva). EGFR was immobilized on a CM5 sensor chip (Cytiva) according to the manufacturer's standard amine coupling protocol at around 1000RU. The VHHs were injected onto the sensor chip at a flow rate of 30 μl/min in two‐fold serial dilutions. The association time was 120 s, and the dissociation time was 300 s. The sensor chip was regenerated at the end of each cycle with two 30 s injections of 10 mM Glycine‐HCl buffer at pH 2.5. The measurements were carried out at 25°C using PBS buffer supplemented with 0.005% Tween20. Data were analyzed using Biacore Insight Evaluation Software (Cytiva).4.4The thermal stability of the VHHs was measured by DSC using a MicroCal PEAQ‐DSC Automated system . The protein samples (1 mg/ml in PBS buffer) were heated from 20°C to 110°C at a scanning rate of 1.0°C/min. The data were analyzed using MicroCal PEAQ‐DSC software .4.5Circular dichroism (CD) spectroscopy measurements were carried out using a JASCO J‐820 spectropolarimeter . For the measurements in the far‐UV region, each of the protein samples  was placed in a 1‐mm quartz cell and measured five times with a bandwidth of 1 nm.2 dmol−1])of each VHH was determined fromθ is the observed ellipticity [deg], C is the concentration of the protein [M], and d is the pathlength [cm].The molar ellipticity  were computed with the GROMACS package  simulations of 7D12 VHH (Unbound state) were performed using GROMACS2016.3e Figure . Five Cα4.7The coordinates and structure factors of 7D12‐Vob and Vobarilizumab have been deposited in the Protein Data Bank with entry codes 7XL1 and 7XL0, respectively.Seisho Kinoshita: Conceptualization ; data curation (lead); investigation (lead); visualization (lead); writing – original draft (lead); writing – review and editing . Makoto Nakakido: Conceptualization (lead); funding acquisition ; project administration ; supervision (lead); writing – original draft ; writing – review and editing . Chinatsu Mori: Data curation ; investigation ; writing – review and editing . Daisuke Kuroda: Conceptualization ; software ; supervision ; writing – review and editing . Jose Caaveiro: Formal analysis ; investigation ; supervision ; writing – review and editing . Kohei Tsumoto: Funding acquisition (lead); project administration (lead); supervision ; writing – review and editing .Appendix S1 The root mean square deviations (RMSDs) of each molecular dynamics simulation with wild‐type 7D12 were shown in Figure Click here for additional data file.DATA S1 7XL0Click here for additional data file.DATA S2 7XL1Click here for additional data file.


During the photochemical relaxation, internal conversion from S1 to T2 takes place, and photochemical hydrogen formation starts on the T2 surface, which passes through a conical intersection, changing to T1. On this triplet surface, the electron that was excited to the Al 3p orbital is transferred to a coordinated water molecule, which dissociates into a hydroxide ion and a hydrogen atom. If the system remains in the triplet state, this hydrogen radical is lost directly. If the system returns to singlet multiplicity, the reaction may be reversed, with recombination with the hydroxide moiety and electron transfer back to aluminium, resulting in water evaporation. Alternatively, the hydrogen radical can attack the intact water molecule, forming molecular hydrogen and aluminium dihydroxide. Photodissociation is observed for up to n=8. Clusters with n=9 or 10 occur exclusively as HAlOH+(H2O)n‐1 and are transparent in the investigated energy range. For n=4–8, a mixture of Al+(H2O)n and HAlOH+(H2O)n‐1 is present in the experiment.Hydrated aluminium cations have been investigated as a photochemical model system with up to ten water molecules by UV action spectroscopy in a Fourier transform ion cyclotron resonance (FT‐ICR) mass spectrometer. Intense photodissociation was observed starting at 4.5 eV for two to eight water molecules with loss of atomic hydrogen, molecular hydrogen and water molecules. Quantum chemical calculations for  A complex sequence of events, including intersystem crossing to the triplet surface and electron transfer, is involved in photochemical hydrogen evolution in Al+(H2O)n. For two water molecules, high‐level calculations reveal a scenario for atomic hydrogen formation in which the system passes through a conical intersection between the two lowest‐lying triplet states, and the hydrogen atom is virtually ejected while the remaining hydrated aluminium hydroxide cation relaxes to its new equilibrium geometry. The lowest‐lying excited state features a half‐filled in‐plane Al 3p orbital in the molecular plane. The minimal overlap between the half‐filled 3p orbital and the water lone pairs slightly favors this configuration over the S2 state with the half‐filled 3p orbital perpendicular to the molecule plane. The third option, a half‐filled 3p orbital aligned along the Al−O bond, causes an unfavorable overlap with the water valence orbitals, and destabilizes the Al−O bond. However, all three molecular orbitals derived from the Al+ [Ne]3s3p configuration have lower excitation energy compared to the atomic ion (7.42 eV),+(H2O) ground state due to an anti‐bonding interaction with the water molecule. For HAlOH+, the two lowest‐lying transitions correspond to excitation from O 2p lone pair orbitals to an empty antibonding σ* molecular orbital with strong Al 3s contribution, at 7.00 and 7.27 eV. Excitation to a π* MO with dominant Al 3p contribution is also possible, starting at 7.84 eV.We start our investigation with the aluminium cation hydrated with a single water molecule, shown in Figure +(H2O)n, n=2–4, as shown in Figure −17 cm2 range for n=2, 3, with somewhat smaller values for n=4. As four stages of nonlinear optics are used to generate tunable laser light in this wavelength region, the pulse energy strongly fluctuates, which leads to the relatively strong scattering of the data points. We therefore provide a running average across five data points to guide the eye. Three fragmentation channels are observed: loss of water molecules, xH2O (red), elimination of atomic hydrogen, H+xH2O (blue), and elimination of molecular hydrogen, H2+xH2O (green), the latter two accompanied by loss of water. For n=2 or 3, loss of a hydrogen radical is the most prominent channel while the formation of a hydrogen molecule becomes competitive for n=4. Pure water loss (red) overall plays a minor role, but exhibits a steep rise at higher photon energies for n=4.Intense absorptions are observed for photodissociation of Al+HOn, n=2+(H2O)n contains one, two and two water molecules in the first solvation shell for n=2, 3, 4. In other words, adding water to the second solvation shell with favorable hydrogen bonds is preferred to Al+ core with three coordinated water molecules. The inserted structure, HAlOH+(H2O)n‐1, is energetically favored for n≥2, and additional hydration leads to increased stabilization. However, CID experiments by Armentrout and co‐workers+(H2O)n structure for n≤4.Quantum chemical calculations reveal that many almost isoenergetic isomers exist, with water molecules in different solvation shells Figure , as repo+(H2O)n‐1 stays transparent in the investigated energy range, thus the observed photodissociation is exclusively due to absorptions of Al+(H2O)n, even though HAlOH+(H2O)n‐1 is thermochemically favored. Still, this does not rule out that a mixture of Al+(H2O)n and HAlOH+(H2O)n‐1 is present in the experiment.Excited state calculations show that HAlOHn=2. The second coordinated water molecule in IIb isomer destabilizes the ground state more than the three lowest‐lying excited states, causing these transitions to shift down to 5.43, 5.97, and 6.26 eV. For HAlOH+(H2O), iIIc, the lowest lying excitations are blue‐shifted to 7.80 and 7.95 eV.The character of the excitations is investigated Figure S1b for n=2. Since the redshift of the 3s–3p excitations strongly depends on the position and orientation of the water molecules interacting with the Al+ 3s and 3p orbitals, the excitations into the lowest excited state differ by up to 0.3 eV between low‐lying isomers. Therefore, very broad bands can be expected in the experiment due to the presence of several isomers at experimental temperatures, consistent with the experimental spectra in Figure +(H2O)n is predominantly present in the experiment. However, the calculated cross‐sections are slightly higher than the experimental ones, up to a factor of ∼3; this may be explained, apart from the error of the computational model, by the presence of transparent HAlOH+(H2O)n‐1 in the experimental mixture. This structure has been reported for n=2 in earlier infrared photodissociation experiments by Inokuchi et al.n=4 could indicate that the transparent HAlOH+(H2O)n‐1 becomes more prominent with increasing cluster size.The calculations provide good agreement with the experimental position and intensity of the bands, although the linearized reflection principle is not able to capture the spectral onset for 2 or H2O on the singlet ground state and lowest‐lying triplet potential energy surfaces (PES) for n=2, see Figure n=1 PES has been studied before.+. However, the exothermic product I3, HAlOH+(H2O) at −0.59 eV, is well separated from I2 by TS2 at 2.05 eV. From I3, H atom loss at 2.85 eV lies well above water loss at 1.41 eV. H2 formation, on the other hand, can now readily proceed via TS4 at 1.07 eV, leading to Al(OH)2++H2 at 0.76 eV. The formation of hydrated aluminium oxide is not competitive, requiring at least 3.64 eV without considering transition states 2 species I1 or I2 while H2 loss with aluminium dihydroxide formation must be expected for HAlOH+(H2O) (I3), with both pathways well separated.We start our investigation of the photochemistry by modeling the observed decomposition involving the loss of H, H2O evaporation with 4.29 eV lies significantly above the barrier for insertion, TS2 at 3.49 eV. From I3, the hydrogen atom is readily lost along a downhill path via TS3, which is placed below I3 by zero‐point correction. Molecular hydrogen formation via TS4, on the other hand, is prohibited, facing a barrier with TS4 at 4.58 eV. Because H2O loss is preferred on the singlet surface and H atom loss seems inevitable in the lowest‐lying triplet state, the experimentally observed formation of molecular hydrogen from Al+(H2O)2 is clearly of photochemical origin. However, photochemistry may also provide pathways for H and H2O loss that differ from this analysis of the singlet ground state and the lowest lying triplet state PESs.On the triplet PES, H1–S3 and T1–T3, on the MRCI/aug‐cc‐pVDZ//BHandHLYP/aug‐cc‐pVDZ level of theory, Figure IIa and IIb, respectively. The triplet states T1–T3 as well as S1 lie energetically lower at I2 than I1, suggesting that the system may relax from the I1 towards the I2 structure upon photoexcitation at I1. Indeed, both minima are interconnected by a transition state TSS1 with energy by 0.12 eV higher than the S1 excitation energy in I1, that is, at 5.86 eV, and can be thus surpassed if enough thermal energy is available .We continue our investigation by an analysis of the first three excited states in singlet and triplet spin multiplicity, S1 state . I2, however, undergoes a significant structural change in S1, leading towards the local minimum I2S1 depicted in Figure 2v symmetry of the cluster. This local S1 minimum lies 0.74 eV below S1 in I2 structure. However, S1 and S0 are still well separated by more than 4 eV, and no conical intersections (CI) between these two states seem to be accessible in the vicinity. Decomposition on the S1 PES can be ruled out on energetic grounds. A multiphoton process seems unlikely, in view of the high intensity observed in the experimental spectra in good agreement with the calculated intensities. Fluorescence from the I2S1 minimum would bring the system to the S0 state in I2S1 geometry, with up to 1.6 eV of relaxation energy available for water evaporation /aug‐cc‐pVDZ level; for BHandHLYP/aug‐cc‐pVDZ, the relaxation energy reaches up to 1.5 eV). However, this energy is not sufficient for hydrogen atom or hydrogen molecule formation. Therefore, fluorescence can be ruled out as the dominant relaxation pathway since preferred loss of atomic hydrogen is experimentally observed.For I1, the minimum in the first excited singlet state does not differ considerably from the Franck Condon point, with the energy lowered by only 0.22 eV through optimization in the S1, which is only accessible after intersystem crossing (ISC). Interestingly, T2 and T3 are almost isoenergetic with S1 in the I2S1 minimum, interpolation coordinate 0.0 in Figure 1–T3 closely correspond to the S1–S3 states with one unpaired electron each in the 3s and 3p orbital of Al, and they lie energetically below their singlet counterparts.The conclusion of Figure +(H2O) minimum I3 in the triplet state. However, I5 will only play a role in the photochemical reaction if it can be reached from I2S1. We therefore performed a linear interpolation between I5 and I2S1 and calculated S0–S3 and T1–T3 single‐point energies along the interpolation coordinate on the MRCI/aug‐cc‐pVDZ level of theory. The resulting curves, Figure 1 indeed undergoes ISC to T2, and the available energy easily suffices to reach the CI between T2 and T1. Passing through this CI, T2 changes to T1 along the interpolation coordinate. The system further relaxes to I5, along a path where T1 and S0 become degenerate, indicating that the hydrogen radical and the unpaired Al 3s electron do not interact significantly.An extensive search of local minima on various excited state potential energy surfaces revealed an interesting biradical structure I5, with a hydrogen radical loosely coordinated to a hydrogen atom of the remaining water molecule, which with 2.80 eV even lies below the HAlOH1 and the two highest‐lying alpha‐spin orbitals in T1, which largely correspond to the unpaired electrons, illustrate how the reaction proceeds mechanistically along the interpolation coordinate . At the IC, interpolation coordinate 0.2, spin density on Al is still ∼1.5, but the dissociating H atom has already acquired spin density of ∼0.5. The spin density of almost exactly 1 at Al and H for interpolation coordinate ≥0.4 indicates that the H atom is fully dissociated, and zero spin density at OH is consistent with a fully developed OH− moiety. The values of the calculated partial charges indicate a similar trend. For technical reasons, CHELPG charge and spin density can only be calculated after the CI, when T1 is the relevant triplet state for the reaction. In the T2 region of the interpolation path, we have to resort to less reliable Mulliken charges. These extrapolate the picture to the I2S1 minimum, and are consistent with the transfer of one electron from the metal center to the water ligand in the 0.0–0.4 region of the interpolation coordinate .Natural transition orbitals in Se Figure b. Around2 formation via TS6 at 3.64 and 3.75 eV, respectively, face appreciable barriers. The energy is available, but statistics disfavors the passage of high‐lying tight transition states. However, if we consider spin crossing back to the singlet surface, both transition states are significantly lowered, becoming almost isoenergetic with the I5 minimum. Therefore, H2O as well as H2 elimination will take place after ISC to the singlet surface while atomic hydrogen formation is preferred in the triplet state.The analysis in Figure S1 minimum and waits to undergo ISC to T2. Passing through a CI to T1 and further relaxation leads to I5, in which an O−H bond is broken and a hydrogen atom is loosely attached to the intact water molecule. The loosely bound hydrogen radical either evaporates directly (the dominant pathway in the experiment) or recombines with OH or attacks H2O on the singlet ground state surface to lose H2O or H2 as observed in the experiment.The analysis of potential photochemical reaction paths in Figures n=3, one may speculate that the photochemical mechanism is analogous, despite the increased absorption intensity at lower photon energies. For n=4, the situation changes significantly, with equal contributions of H and H2 loss while water loss becomes significant only at photon energies above 5.2 eV. With the redshift of the transitions and the increased conformational flexibility of the larger cluster, more photochemical reaction pathways will become available. Photodynamics is sensitive to the excited state reached after excitation, and the intriguing behavior of the n=4 branching ratio indeed suggests that an S1/S0 CI to the ground state may become accessible at higher photon energies, but this is only a speculation.Because the experimental product branching ratio is very similar for The photochemical decomposition can be expected to be extremely complicated in detail and heavily dependent on the cluster structure, exact energetic location of the available ISCs, ICs and transition states. In any case, a more quantitative picture would require molecular photodynamics employing multi‐reference methods including spin flips,n>4 was only studied experimentally for cluster sizes n=5–10 n rearranges to HAlOH+(H2O)n‐1, and we observe the Al−H stretching mode in our infrared spectra in traces for n=4, with quickly increasing intensity for larger clusters.+(H2O)n‐1 does not absorb UV light with λ≥225 nm for n=2–4, and hydration does not seem to lead to an appreciable redshift. We therefore assign the size‐dependence of the absorption cross‐section and the disappearance of the absorption to the presence of the HAlOH+(H2O)n‐1 structure. As the intensities fluctuate, we conclude that the conversion of Al+(H2O)n into HAlOH+(H2O)n‐1 due to its exothermicity[n=8 or 9, assuming that at least four water molecules evaporate. At T≈100 K, interconversion of Al+(H2O)n is obviously slow for n≤8 since these species exhibit intense UV absorptions on the timescale of the experiment, which is several seconds. However, n=9 is almost completely converted, indicating that this cluster size readily interconverts under the experimental conditions, and only a small fraction of Al+(H2O)n remains.It is well established that Aln>4 is assigned to an asymmetric solvation of the Al+ core,+ remains doubly coordinated for n≤8.The absence of further redshift for n=5–8, water loss becomes the dominant decomposition channel towards higher photon energies. Hydrogen molecule formation is observed in all four cases. H atom loss, however, is suppressed for n=5 and n=8 while it occurs at competitive intensities for n=6 or 7. For n=7, it is even the dominant photochemical reaction path below 4.8 eV, but disappears around 5.0 eV. As discussed above, increasing solvation statistically favors water evaporation. Water loss can occur, for example, after excitation followed by fluorescence. This scenario is supported by the experimental trend towards higher photon energies where only one or two water molecules are lost while the photon energy is sufficient for evaporation of almost the complete hydration shell. Accessibility of IC or ISC depends sensitively on cluster structure, and a single water molecule might lead to significant changes in photochemical behavior. One factor in the suppression of the H atom loss channel is the integration of the first shell water molecules into the hydrogen‐bonded network, which reduces their conformational flexibility. However, this also applies to the H2 elimination pathways, which are still active for n=8. Unfortunately, quantum chemical analysis of the photochemistry becomes difficult with increasing cluster size, with each water molecule adding nine dimensions to the potential energy surface of the cluster.For the photochemical decomposition of +(H2O)n to HAlOH+(H2O)n‐1 in hydrated aluminium cations with increasing cluster size, and for n=9 or 10, only the inserted structure is present, as evidenced by the lack of measurable absorption cross‐section for wavelengths above 225 nm. While black‐body infrared radiative dissociation (BIRD) triggers molecular hydrogen formation from HAlOH+(H2O)n‐1, photochemical reaction pathways lead to the evolution of atomic as well as molecular hydrogen from Al+(H2O)n. For n≤4, the formation of H atoms is preferred over molecular hydrogen elimination. Quantum chemical analysis reveals that a singlet–triplet intersystem crossing is required to explain all observed photochemical reaction products. For n=2, the Al 3p electron in the S1 state is transferred to a σ* orbital of a water ligand; this weakens the O−H bond and ultimately leads to formation of atomic hydrogen and a hydroxide ligand. Before its final release, the roaming H atom might abstract hydrogen from the intact water ligand, thus explaining H2 formation, or recombine with the hydroxide with concomitant electron transfer back to the metal center, essentially reversing the reaction. The accumulated relaxation energy in the cluster triggers water evaporation in the electronic ground state. Photochemical hydrogen evolution in hydrated aluminium is thus fundamentally different from the known ground‐state reaction induced by BIRD, and does not involve a HAlOH+(H2O)n‐1 hydride‐hydroxide structure.Photodissociation spectroscopy confirms the transition from AlThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. 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