Despite the bulk of the carbazolyl moiety, the silylenylium cation [RSi]+ retains high reactivity. It was shown to react with an amine to form three bonds at the silicon atom in one reaction which conforms with the notion of a “supersilylene”. The resulting silylium cation [RSi(H)NR′2]+  obtained by oxidative addition of an NH bond at [RSi]+ is even more acidic than the silylenylium cation (SiII) due to the absence of a lone pair of electrons the silicon atom.Mono‐coordinated silicon(II) cations are predicted to be reactive ambiphiles, combining the typically high Lewis acidity of silicon cations with nucleophilicity due to the presence of an electron pair at the same atomic centre. Here, a carbazole‐derived scaffold was used to isolate salts with a mono‐coordinated silicon(II) cation, [RSi] A mono‐coordinated silicon cation was prepared by halide abstraction from a iodosilylene. It is shown to react with an amine to form three bonds at the silicon atom in one reaction which conforms with the notion of a “supersilylene”. West's landmark report detailing the isolation of the first silylene in 1994 initiated the investigation of their reactivity.2 and SiBr2 can be stabilized by suitable carbene donors to make molecular synthetic equivalents of SiII available.2, C2H2, NH3, or CO2 as shown by Aldridge,Silylium cations  in the gas phase indicated that three bonds are formed in a single reactive encounter which prompted the authors to coin the phrase “supersilylenes” for these cations.II cations with a higher coordination number could be obtained, such as Jutzi's [Cp*Si]+ and masked SiII cations of the general formula [R′Si(L)x]+ .II cation benzene had been intermediarily present.+).A combination of enhanced Lewis acidity and ambiphilicity was predicted for cationic SiR=1,8‐bis‐3,6‐di‐tert‐butyl‐carbazolyl) in our group, the question whether analogous silicon compounds were accessible as well, inevitably had to be asked. Initially the issue was tackled computationally  is less stable than the chlorosilylene isomer by 25.7 kJ mol−1. On the other hand, the CH insertion product (isomer II) is thermodynamically favoured over the chlorosilylene RSiCl, but the activation barrier of 202.5 kJ mol−1 for this reaction is too high to be feasible. Similar energy profiles were found for the other base‐free halosilylenes RSiX  and the silylenylium cation [RSi]+ (details see SI 4.1). With these data available, experiments were initiated.After the recent synthesis of heavier chlorotetrylenes and the corresponding tetrylenium cations bearing a sterically demanding carbazolyl substituent . The molecular structure of these trihalosilane derivatives is surprising in the regard that the N−Si bond is bent out of the plane spanned by the carbazole C atoms by more than 40° (see SI 2.1–2.3), thereby reducing the effective steric bulk of the carbazolyl substituent drastically. The 29Si NMR resonances were found in the expected range for the respective trihalosilane . While all attempts of reduction of RSiCl3 to RSiCl failed, the heavier homologues RSiBr3 and RSiI3 could be reduced to afford the first base‐free halosilylenes, RSiBr (2Br) and RSiI (2I), where the best results were achieved with MgI compounds as reducing agents.3 and [(MesBDI)Mg]2  in toluene. After sonication at ambient temperature, the solvent was evaporated and the solid was extracted with n‐hexane to afford RSiX in moderate yields . By removal of two substituents from the silicon atom the occupied space is reduced, hence the molecular structures show the silicon atom in the carbazole plane =+147.5 ppm). The compound could be isolated and identified as the bis‐carbazolyl silylene R2Si which is the bulkiest silylene known to date (see SI 2.5.1).The metathesis reaction of potassium carbazolide (R‐K) with silicon halides SiX° see SI .1–2.3, t2Br and 2I could prove versatile starting materials for a wide range of reactions, but the focus of this study was set on halide abstraction reactions. Due to the high inherent reactivity of the silylenes and the reaction product, viable solvents for these reactions were only hydrocarbons and aromatic solvents. As a halide abstraction reagent, Ag+ salts were chosen, as neither Li+, Na+, K+ or Cs+ salts showed any reactivity in these solvents. Initial experiments of the reaction of 2Br with Ag[wca] [wca=Al(OC4F9)4] yielded an intractable mixture of compounds in which no [RSi]+ was detected. However, the reaction of 2I with Ag[wca] afforded [RSi][wca] (3) in 83 % yield as a yellow compound. In solution, the cation was characterized by multinuclear NMR spectroscopy in excellent agreement with the computationally predicted values for donor‐free [RSi]+ in the gas phase. The 1H NMR spectrum clearly indicates a Cv2 symmetric compound as all CH3 groups of the arene‐tert‐butyl moieties are magnetically equivalent. In the 13C NMR spectrum, there is a characteristic shift of the resonance of the meta‐C atoms of the arenes to higher frequencies (164.26 ppm) which was also observed for the heavier homologuous [RE]+ species .15N NMR resonance  is significantly shifted compared to the starting material, RSiI . In contrast, 29Si NMR spectroscopy revealed that the Si atom is more shielded in 3  than in 2I . This shielding effect can be rationalized by considering the interaction with the flanking arenes: removal of one of the flanking arenes in the computed model compounds does not cause a significant shift in the 29Si NMR resonance (δ(29Si)=+75 ppm), but upon removal of both arenes, the resonance is predicted at δ(29Si)=+604 ppm (see SI 4.2). These values are in stark contrast to the value found for the penta‐coordinated [Cp*Si]+ (δ(29Si)=−400 ppm).3 provides a stabilization energy of 181.2 kJ mol−1 (see SI 4.6). After many attempts, crystallisation by slow concentration of a solution of [RSi][wca] in fluorobenzene was successful, allowing the unambiguous identification of [RSi][wca] as a salt in the solid state  Å) and even the SiIV compound RSiI3 (1.770(2) Å). The three shortest Si‐C contacts to the flanking arenes in [RSi]+ are 2.654(2), 2.732(2) and 2.741(2) Å which is considerably longer than in Jutzi's [Cp*Si]+ (2.14 to 2.16 Å) and typical silylium‐arene contacts in arene adducts of [Et3Si]+ (2.18 Å) and [Me3Si]+ (2.12–2.18 Å).The halosilylenes Si][wca]  in 83 % ϵ=2.33×103 L mol−1 cm−1) is accurately reproduced by computations . As it is the lowest energy electronic transition, HOMO and LUMO are the major contributors for this electronic excitation ]+ (4) to the isomeric silylium ion [RSi(H)NHtBu]+ (5). However, when the experiment was conducted and studied by NMR spectroscopy, a spin system consistent with another product was observed which was assigned to the amine adduct of the silylium cation [RSi(H)(NHtBu)NH2tBu]+ (6). In good agreement with the computed value, the 29Si NMR resonance was observed at −29.4 ppm . The 1H NMR spectrum indicates the presence of two distinct NtBu groups in proximity to the SiH moiety as well as the presence of an NH and an NH2 moiety (see SI 2.7). No intermediate was observed and no reaction to 5 occurred after addition of another equivalent of [RSi][wca] . The reaction was then studied in silico, confirming that for [RSi]+ the reaction with an amine in solution is exergonic and likely to proceed stepwise ]+ (4) should be formed initially (ΔG=−74.6 kJ mol−1) which then undergoes a proton shift reaction (ΔG=−42.8 kJ mol−1) to the isomeric silylium cation [RSi(H)NHtBu)]+ (5). It should be noted that the monomolecular rearrangement reaction form [RSi(NH2tBu)]+ (4) to [RSi(H)NHtBu]+ (5) has a large activation barrier of 206.4 kJ mol−1 (computed with a reduced model for both the carbazole and the amine) which is thermally unattainable, so it is likely to proceed with additional molecules of the amine as proton shuttle. Then, the adduct formation with another molecule tBuNH2 to give 6 (ΔG=−113.5 kJ mol−1) is more exergonic than the first step forming 4 (see SI 4.4). This indicates a fascinating feature of the silylenylium cation [RSi]+, as for this strong Lewis acid, oxidative addition of the NH bond of a substrate molecule yields a silylium cation which is an even stronger Lewis acid. This is corroborated by the computed affinities for fluoride  and hydride ions  which are lower for the silylenylium ion [RSi]+ than derived silylium ion 5 (see SI 4.3). Also, the computed charges of the silicon atom indicate this tendency, as in [RSi(H)NHtBu]+ the silicon atom bears a considerably more positive charge than in [RSi]+ . The reaction of silylenylium cations with suitable substrates such as amines enables a new route to H‐substituted functionalized silylium cations, the first isolated examples of which have only been reported very recently by Oestreich.With the silylenylium salt )NHtBu]+ . However+ by halide abstraction from the first donor‐free halosilylenes is presented. Stabilized by a carbazole scaffold and arene interactions, the cation bears no σ‐donors except for the lone substituent. The predicted reactivity for this type of cation leading to the formation of three bonds at the silicon atom could be demonstrated by a reaction with an amine, enabling a new route to silylium cations bearing an H substituent and a functional group.In conclusion, the synthesis of the mono‐coordinated silylenylium cation [RSi]1Cl), 2010373 (1Br), 2010374 (1I), 2010375 (2Br), 2010376 (2I), 2010377 (3), 2010378 (2SiR) and 2010379 (2I‐II)2010372  should be addressed to the authors.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.


Scientific Reports 10.1038/s41598-018-37304-0, published online 30 January 2019Correction to: 2)” or “TgSO2” for simplification. As such, in the “Results and Discussions” section:This Article contains a repeated typographical error, where the wrong unit has been used for the emissions of sulfur. In this Article, the unit “TgS” has been used instead of the correct unit “TgSOx (as SO2 emissions were reduced by 55 TgS (31%) from 1990 to 2015. Individual regions have had different contributions to the global emission budget throughout this period  as also documented in other studies1,14,21,25,37. The largest decrease in global SO2 emissions occurred in the first decade, from 1990–2000 and was mainly due to a large reductions in Europe (−42 TgS/−54%). There was a smaller decrease in North America (−7 TgS/−21%) during this time, and an increase in East Asia (+10 TgS/32%). In comparison, in the following period 2000–2015, emissions in Europe and the US decreased by a similar total amount (−14 and −13 TgS) or in relative terms, respectively by −40% and −50%. In Eastern Asia, there was an increase of the emissions up to 2005 by more than +20 TgS (70%), while in the last ten years from 2005 to 2015 there has been a reduction, we have used emission inventories with a decrease of −6 TgS (−13%). For the whole 25 year period from 1990 to 2015, India’s emissions increased from 4.5 to 15 TgS, while in Africa only small changes occurred, +1 TgS (8%).”“Globally the SOshould read:2 emissions were reduced by 55 TgSO2 (31%) from 1990 to 2015. Individual regions have had different contributions to the global emission budget throughout this period  as also documented in other studies1,14,21,25,37. The largest decrease in global SO2 emissions occurred in the first decade, from 1990–2000 and was mainly due to a large reductions in Europe (−42 TgSO2/−54%). There was a smaller decrease in North America (−7 TgSO2/−21%) during this time, and an increase in East Asia (+10 TgSO2/32%). In comparison, in the following period 2000–2015, emissions in Europe and the US decreased by a similar total amount (−14 and −13 TgSO2) or in relative terms, respectively by −40% and −50%. In Eastern Asia, there was an increase of the emissions up to 2005 by more than +20 TgSO2 (70%), while in the last ten years from 2005 to 2015 there has been a reduction, we have used emission inventories with a decrease of −6 TgSO2 (−13%). For the whole 25 year period from 1990 to 2015, India’s emissions increased from 4.5 to 15 TgSO2, while in Africa only small changes occurred, +1 TgSO2 (8%).”“Globally the SOAdditionally,18,20; (shown as ‘Alternative emissions’).”“To illustrate the difference between the emissions used by the models in this study and the most recent estimates for the last ten year period in East Asia, the new inventories are included in Fig. 1, showing a decrease of −18 TgS (−34%) between 2005–2015should read:2 (−34%) between 2005–201518,20; (shown as ‘Alternative emissions’).”“To illustrate the difference between the emissions used by the models in this study and the most recent estimates for the last ten year period in East Asia, the new inventories are included in Fig. 1, showing a decrease of −18 TgSOFinally, the incorrect unit is also displayed in Figure 1 and Figure S3. The correct Figures 1 and Figure S3 are displayed as Figures


MERS‐CoV), but other livestock species  are also susceptible to infection with MERS‐CoV. Animal‐to‐animal transmission in alpacas was reported, but evidence for transmission in other species has not been proved. This study explored pig‐to‐pig MERS‐CoV transmission experimentally. Virus was present in nasal swabs of infected animals, and limited amounts of viral RNA, but no infectious virus were detected in the direct contact pigs. No virus was detected in the indirect contact group. Furthermore, direct and indirect contact pigs did not develop specific antibodies against MERS‐CoV. Therefore, the role of pigs as reservoir is probably negligible, although it deserves further confirmation.Dromedary camels are the main reservoir of Middle East respiratory syndrome coronavirus ( Three extra animals were used as negative controls . Animals from G1, G2 and G3 were housed in the same experimental box unit but placed in two different pens. The pens were separated by two fences with a 30 cm distance among them  MERS‐CoV (passage 7 human isolate HCoV‐EMC/2012) in 3 mL saline solution via intranasal route (1.5 mL in each nostril). Two days later, all tarpaulins were removed and G2 pigs were moved from pen 2 to pen 1 until the end of the study.G1 was inoculated with 10All animals were monitored daily for clinical signs , as well as rectal temperatures until day 10 post‐inoculation (PI). Nasal swabs (NS) were obtained on days 0, 1, 2, 3, 4 PI from all animals and at days 7, and 10 PI from G1. Animals from G2 and G3 were also sampled at 5, 6, 9 and 12 days PI, corresponding to days 3, 4, 7 and 10 after direct (G2) and indirect (G3) contact with G1.2.2Two independent NS were collected and placed in PBS  and DMEM containing antimicrobial drugs (for detection of infectious virus); swabbing was performed deep in both sides of the nasal cavity. Sera were obtained before challenge and at 7, 15 and 26 days PI, and they were subsequently used to detect the presence of MERS‐CoV‐specific antibodies. Negative control pigs were sampled (NS and sera) and euthanized before the start of the experiment.Daily environmental samples (ES) between day 0 and 10 PI were obtained from air sampling and wall surface swabbing  following the manufacturer's instructions. The RNA extracts were tested by the UpE PCR (Raj et al., Viral RNA from NS and ES was extracted with NucleoSpin2.4Serum samples from days 0, 7, 15 and 26 PI were tested to determine the specific S1‐antibodies by a MERS‐CoV S1‐ELISA, and by a specific virus neutralization assay, as previously described (Haagmans et al., 3Similar to a previous experiment (Vergara‐Alert, van den Brand, et al., Other livestock besides dromedaries are susceptible to MERS‐CoV infection (Crameri et al., Suidae in the transmission of MERS‐CoV should be further clarified, unlike camelids, pigs do not seem to be able to transmit MERS‐CoV efficiently, suggesting that the role of pigs as reservoir is probably negligible.In summary, using our pig model of MERS‐CoV infection (Vergara‐Alert, van den Brand, et al.,


Besides known [(PhSi)4S6] (IV), new compounds [(NpSi)4S6] (1) and [(StySi)4S6] (2) were obtained, the first two of which underwent reactions with [AuCl(PPh3)] to form ternary complexes. DFT studies of cluster dimers helped us understand the differences between the habit of {Si4S6}‐ and {Sn4S6}‐based compounds. Crystalline 1 showed a pronounced nonlinear optical response, while for intrinsically amorphous 2, the chemical damage threshold seems to inhibit a corresponding observation. Calculations within the independent particle approximation served to rationalize and compare electronic and optical excitations of [(RSi)4S6] clusters . The calculations reproduced the measured data and allowed for the interpretation of the main spectroscopic features.We report the extension of the class of organotetrel sulfide clusters with further examples of the still rare silicon‐based species, synthesized from RSiCl 4S6] , and their products upon reaction with [AuCl(PPh3)], [{RSi(μ‐S)}2{AuPPh3(μ‐S)}2]. Quantum chemical studies of cluster dimers elucidated the differences in the habit of silicon and tin compounds, and DFT calculations of crystalline models confirmed the applicability of these methods.To gain insight into the nonlinear optical properties and reactivity of organotetrel chalcogenide clusters, we studied the silicon homologues, [(RSi) The chemical and structural properties of tetrel chalcogenide clusters with organic substituents have been extensively studied in the past.4S6] (Sty=4‐vinylphenyl), which was determined via DFT calculations to have a heteroadamantane‐type molecular structure, was recently shown to possess an extreme nonlinear optical behavior.4S6] with cyclic and/or aromatic substituents R to understand how the substituents influence the compounds’ properties. Generally, it was shown that distinct order within the solid material  leads to second‐harmonic generation (SHG), while a high degree of amorphousness results in white‐light generation (WLG).In addition to the chemical features of such clusters, the styryl‐substituted cluster  (IV) we herein report on the successful synthesis of two new organosilicon sulfide compounds, [(NpSi)4S6] (1) and [(StySi)4S6] (2), their characterization and follow‐up chemistry with gold complexes to form ternary complexes [{RSi(μ‐S)}2{AuPPh3(μ‐S)}2] . We used density functional theory (DFT) methods to gain insight in structural features of the adamantane‐based compounds, in particular regarding inter‐cluster interactions that contribute to the macroscopic habitus of the solid. All new compounds were investigated regarding their linear and nonlinear optical properties, which were additionally studied by means of quantum chemistry. The calculations explain the preferred habitus of the compounds, and reveal the deep impact of the ligands in the optical responseThe preparation of corresponding silicon analogs from RSiCl3 with R=phenyl , naphthyl , and styryl  with Na2S discussed herein are summarized in Scheme The reactions of RSiCl3 (II) as a precursor for [(NpSi)4S6] (1), single crystals were obtained upon distillation, and the molecular structure 4S6] , single pSi)4S6] , single II with 1.5 equivalents of Na2S affords a crystalline, colorless solid. Upon dissolution in toluene and cooling to −25 °C, single crystals of [(NpSi)4S6]⋅0.5 C7H8 (1⋅0.5 C7H8) suitable for X‐ray diffraction were obtained. The molecular structure of 1 is shown in Figure 1 crystallizes in the triclinic space group P1‾with two molecules per asymmetric unit. Like [(PhSi)4S6], 1 is an adamantane‐type Si/S cluster with four organic substituents, each bound to one Si atom. The molecule shows minor deviations from dT symmetry, due to slightly different angles in the inorganic core and different orientations of the organic substituents. Furthermore, the two individuals differ greatly in the orientation of the organic substituents 4S6] are amorphous ,The reaction of 3 (III) with 1.5 equivalents of Na2S should proceed similarly and lead to the formation of [(StySi)4S6] (2). However, only the crude product could be isolated upon evaporation of the solvent in form of a colorless “solid” with the consistency of cotton candy or honey—depending on the duration of the reaction and drying process. We attribute this to the high polymerization tendency of the styryl substituents, and the corresponding oligomerization/polymerization that proceeds visibly as time goes by. Notably, this was not the case for the Sn analogs, which we take as a further hint for the correlation of the nature of the T/E cluster core with the properties of the organic substituents: in the Sn/S clusters, the styryl substituents of neighboring cluster units are obviously not close enough to allow for a chemical reaction of neighboring vinyl groups. As a consequence of the different situation with an underlying Si/S cluster core, 2 could neither be obtained as single crystals, nor could individual clusters be detected by means of liquid injection field desorption/ionization (LIFDI) or electrospray ionization (ESI) mass spectrometry. However, we were able to collect 29Si NMR data of a freshly prepared sample of 2 that turned out to be sufficiently soluble. A downfield shift of ≈9 ppm of the observed singlet with respect to the precursor signal is in accordance with the NMR data of other Si/S adamantane‐type clusters and their corresponding organosilicon trichloride precursors reported previously, such as [(EtSi)4S6]/EtSiCl3, [(PhSi)4S6]/PhSiCl3, or [(CH2=CHSi)4S6]/CH2=CHSiCl3.1H and 13C NMR spectra, which clearly show the signals of the styryl substituent at a significant downfield‐shift compared to the starting compound III, with the ipso‐ and meta‐atoms being the most affected, which indicates a reaction at the Si atom to have taken place. Hence, the NMR data indicate the formation of a compound comprising individual adamantane‐type clusters [(StySi)4S6]. Yet we assume that they start to unstoppably form oligo‐/polymers shortly upon synthesis.The reaction of StySiCl2S and [AuCl(PPh3)] leads to the fragmentation of the clusters and the formation of the four‐membered rings [{RSi(μ‐S)}2{AuPPh3(μ‐S)}2] . Colorless single crystals of 3⋅CH2Cl2 and 4 were obtained by layering the respective reaction solutions with n‐hexane. Their molecular structures are shown in Figure 3⋅CH2Cl2 crystallizes in the monoclinic space group C2/c, and 4 crystallizes in the triclinic space group P1‾, both with half a molecule per asymmetric unit. The molecules in these compounds have an inversion center in the center of an {RSi(μ‐S)}2 ring, with nearly linear {Au(PPh3)(μ‐S)} substituents attached to the Si atoms. Although the structural parameters of the central unit in both compounds are quite similar, they differ significantly in the orientation of the {Au(PPh3)(μ‐S)} units: whereas they point away from the central {Si2S2} ring in 3 with a C1−Si1−S2−Au1 cis arrangement, the corresponding atoms in 4 show a trans arrangement. Thus, while the interatomic distances in these compounds are similar, the Si1−S2−Au1 and S2−Au1−P1 bond angles are different. Homologous Sn/S compounds exhibiting this structural motif as well as a cis or trans arrangement depending on the organic moiety at the tetrel atom were described previously, showing the same trends in the T−S−Au and S−Au−P bond angles.Further studies on the reactivity of the adamantane‐like organosiliconsulfide clusters were undertaken. The addition of Na [AuClPPh] leads t4, a further downfield shift by 5.6 ppm was observed in the 29Si NMR spectrum. Table I–IV, 1, 2, and 4. Note that NMR data of compound 3 were not obtained owing to very poor solubility of the crystals.Upon fragmentation of the adamantane‐type cluster and formation of 4S6] (IV) and [(NpSi)4S6] (1) are observed in a crystalline phase, while [(PhSn)4S6] and [(NpSn)4S6] are intrinsically amorphous. Nevertheless, it was suggested that [(NpSn)4S6] features a structure with a higher amount of order compared to [(PhSn)4S6], owing to more efficient π‐stacking interactions between the Np substituents as compared to the Ph substituents.It was experimentally shown, that [(PhSi)4S6] and [(RSi)4S6] cluster compounds with R=Ph, Np, one may consider the smaller radius of the {Si4S6} core with respect to the {Sn4S6} core to enable a stronger interaction of the neighboring clusters through more efficient π‐stacking interactions. To investigate this hypothesis, quantum chemical calculations of the interaction in cluster dimers as a minimal model were carried out, which already provide valuable insights into the cluster interaction that can be transferred to the extended crystalline or amorphous material.To explain the general difference observed in the  cluster and 9 kJ mol−1 for the [(NpSn)4S6] cluster  in comparison with the corresponding Sn−C distance (2.088 Å) increases the barrier by a stronger interaction between the hydrogen atom of the substituent and the sulfur atoms of the cluster. The relative positions of S and H atoms at the rotation barrier are visualized in Figure In order to classify the flexibility of the substituents, we initially calculated the rotation barriers of the substituents for both cluster cores. Rotation barriers of the substituent at the single cluster are hardly present  than for those with smaller core radius (Si/S), which is in agreement with the tendency for (a) higher order in compounds with R=Np than in compounds with R=Ph, and (b) higher order in {SiThe investigation of the cluster dimer structures was performed to analyze the interaction between the molecules with different cluster cores and substituents. The cluster dimer structures were determined by two computational approaches and their results were combined to obtain a larger number of possible conformers. The dissociation energies of all cluster dimers are plotted against their core–core distance in Figure 4S6] cluster dimers show a core–core distance range that is shifted to slightly smaller values than the range calculated for [(NpSi)4S6]. The same trend, but even more pronounced, is observed for the {Sn4S6}‐based cluster dimers. Cluster dimers in this region of inter‐cluster distances yielded stacked or alternating substituents and different orientations of the cluster cores  is much larger (by 0.5 to 1.0 Å) as compared to the calculated cluster dimers, as in the crystal, the interaction is shared between more than two clusters. Besides the majority of cluster dimers with a core–core distance between 6.0 and 6.5 Å, there are a few cluster dimers with core–core distances between 7.2 and 8.0 Å, with a poor relative orientation of the clusters to each other 4S6]. A similar, but slightly smaller, difference in dissociation energies is calculated for the corresponding {Sn4S6}‐based cluster dimers (≈53 kJ mol−1). Obviously, the larger Np substituents lead to a stronger interaction between cluster dimers than Ph substituents. Other than originally anticipated, the investigation of the cluster dimers did not indicate larger dispersive interaction for clusters comprising a smaller radius of the cluster core. The {Sn4S6}‐based clusters have a higher absolute dissociation energy than the {Si4S6}‐based cluster dimers, which illustrates a stronger interaction overall. To understand this result, we performed a decomposition analysis of the cluster dimer binding energy contributions into the substituent–substituent interaction, the substituent–core and core–core interaction 4S6]. We therefore believe that these insights can be transferred to crystalline and amorphous cluster materials with core–core distances between the medium and large core–core distance regions of Figure The decomposition of the binding energy reveals similar substituent–substituent binding energies for cluster dimers with the same substituent. The larger naphthyl substituents show a higher binding energy than the smaller phenyl substituents due to larger dispersive interactions. In contrast, the core–core binding energy is larger for the Sn/S‐based cluster dimers than for the Si/S based cluster dimers due to the larger adamantane core. This stronger core–core interaction for the larger Sn/S‐based cluster dimers leads to higher absolute values of the dissociation energies than observed for the Si/S‐based analogs. For the cluster dimers with larger core–core distances 4S6] the relatively similar strengths of core–core and substituent–substituent interactions allow for a higher degree of intermolecular order, which is in agreement with the  experimental findings: the powder produces SHG (as a sign of phase matching) in spite of an apparently amorphous nature according to X‐ray diffraction. The results of this study thus contribute significantly to the overall understanding of the origin of (dis)order in compounds of the type [(RT)4E6] .In conclusion, we propose that the predominance of the relatively isotropic core–core interactions in  and [(NpSi)4S6]. The structural relaxation of single clusters and molecular crystals performed by the plane‐wave implementation of the DFT within the periodic supercell method leads to geometries of the same symmetry and in overall close agreement with the structures calculated with a localized basis. The optimized geometries were employed for the calculation of the electronic and optical excitations.We investigated the role of the substituents (phenyl vs. naphthyl) on the optical response of the molecular clusters. Thereby, we modeled two of the synthesized clusters with the same core but different substituents, [(PhSi)4S6] and [(NpSi)4S6] clusters. The structural differences between the cluster cores of the two molecules are limited. The Si−S distance amounts to 2.15 Å in both clusters, while the S−C bond length is 1.87 Å in [(PhSi)4S6] and 1.88 Å in [(NpSi)4S6]. However, the different substituents have a deeper impact on the molecular electronic structure. The (degenerate) HOMO of the [(PhSi)4S6] molecule is localized at the S atoms in the cluster core, while the HOMO of the [(NpSi)4S6] cluster is localized on the naphthyl rings. The LUMO of both systems is similar and localized on the substituents 4S6] cluster. Quasiparticle effects, calculated in an approximate manner by the ΔSCF method as described in Ref. 4S6] and 5.09 eV for the [(NpSi)4S6] cluster . The energy of the lowest excitonic excitation, describing the transition of one electron to the LUMO leaving a hole behind in the HOMO, is calculated following the procedure described in Ref. 4S6] and 2.89 eV for the [(NpSi)4S6] cluster, respectively, are predicted. These energies are rather close to the HOMO–LUMO gap, suggesting that the quasiparticle shifts are nearly canceled out by the electron‐hole attraction. Thus, as many‐body effects due to the electron‐electron and the electron‐hole interaction counterbalance, the IPA‐calculated optical excitation spectra are expected to yield a reasonable description of the measured optical response, at least for the low‐energy excitations.Figure 4S6] (blue line) and [(NpSi)4S6] (black line) single clusters is shown in Figure The extinction coefficient of [(PhSi)4S6] cluster (black line). Structures in the first peak match quite well the energy of the HOMO–LUMO+1,2 (degenerate) and HOMO–LUMO+3. The main peak at about 4 eV is mainly due to transitions from the HOMO to a group of energetically close states localized at the C atoms of the naphthyl rings. Instead, in the case of the [(PhSi)4S6] cluster (blue line), the onset of the optical absorption is located at much higher energies than the DFT‐calculated HOMO–LUMO difference. Indeed, the HOMO–LUMO transition probability is small, due to the different spatial localization and orbital character of the two states. We assign the first spectral peak to transitions from the HOMO to three molecular orbitals about 4.4 eV above the HOMO and with a spatial extension partially overlapping the HOMO. Summarizing, the absorption spectra of [(PhSi)4S6] and [(NpSi)4S6] clusters are rather different concerning the positions and line shapes of the spectral features. The deviations in the optical response originate from the qualitatively different and differently localized electronic states involved in the transitions resulting in the main spectral peaks of the two compounds.The onset of the optical absorption corresponds exactly to the DFT‐calculated HOMO–LUMO transition in the case of the  (1) comprises toluene molecules . As illustrated in Figure S17, the extinction coefficient calculated with and without toluene does not substantially differ, demonstrating that the optical response of the molecular cluster is not affected by the presence of solvent. The extinction coefficient calculated within the IPA for the [(PhSi)4S6] (IV) derived and [(NpSi)4S6] (1) derived molecular crystals is shown in Figure We then proceeded with the optical characterization of the synthesized crystalline compounds. Owing to the synthesis method, the experimental crystal structure of  cluster (1) with the measured data, we accounted for many‐body effects in an approximated manner by means of a scissors‐shift.In order to compare the calculated spectra of the crystalline material comprising  (IV) and [(NpSi)4S6] (1) shows vanishing optical nonlinearities for all tensor components, as expected for a centrosymmetric crystal structure. Deviations from this behavior in experiments thus arise from structural defects and amorphous regions in the samples, as well as from surface‐related contributions. At least for centrosymmetric crystals, they are not inherent bulk properties.The calculation of the second‐order polarizability tensor for the crystalline phases of [(PhSi)4S6]. We added new members with R=naphthyl  and styryl , which were investigated with respect to their reactivity towards [AuCl(PPh3)], their structures, and their optical properties by a combination of comprehensive experimental and theoretical studies. The investigations contributed to characterize and further understand the chemical behavior of such clusters, which showed to behave differently from the much better known germanium and tin homologues. Our studies indicate a much more pronounced tendency to polymerization in case of 2 than observed for its Sn homologue, and confirm the expected, much higher air and water sensitivity of the Si compounds. Second, the observation that clusters with the combination Ph/Si/S (reported previously) and Np/Si/S (1) are crystalline, while the Sn homologues are amorphous was studied on the bases of cluster dimer models calculated with DFT methods. Different types of intra‐cluster interactions were found, that led to different dissociation energies as a function of the nature of the substituents, their relative orientation, and the core compositions {Si4S6} versus {Sn4S6}. The detailed study ultimately helped to explain the experimental findings on the basis of much stronger isotropic interactions in the {Sn4S6}‐based cases in comparison with directional interactions that play a much more notable role in the {Si4S6}‐based materials. Regarding the optical properties of the new compounds and the products of their reaction with the gold complex, we could show that the emission is quenched upon fragmentation and formation of the gold compounds. Moreover, the crystalline compound 1 was proved to exhibit SHG as a consequence of high order that allows for phase matching; however, the SHG signal does not stem from the crystalline bulk but should be attributed to surface phenomena or structural defects. The behavior of 2 is clearly different, but its chemical nature inhibited a statement regarding the compound's nonlinear response. The experimental findings were corroborated by static and time‐dependent DFT calculations of both the molecules and the crystalline material, which are in full agreement with the measurements and therefore indicate the applicability of the used methods and models.We presented the synthesis of new members of the rare class of organosilicon sulfide clusters of the general formula [(RSi)The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Accordingly, the cluster with the lowest proportion of CD8 T cells, M1 Macrophages and highest proportion of M0 Macrophages has the worst outcome among clusters. By grouping patients with similar immune patterns, we are also able to suggest treatments that are specific to the tumor microenvironment.Tumor immune microenvironment has been shown to be important in predicting the tumor progression and the outcome of treatments. This work aims to identify different immune patterns in osteosarcoma and their clinical characteristics. We use the latest and best performing deconvolution method, CIBERSORTx, to obtain the relative abundance of 22 immune cells. Then we cluster patients based on their estimated immune abundance and study the characteristics of these clusters, along with the relationship between immune infiltration and outcome of patients. We find that abundance of CD8 T cells, NK cells and M1 Macrophages have a positive association with prognosis, while abundance of  It usually starts in the femur, the tibia and the humerus, and less commonly the skull, the jaw or the pelvis . There aAlthough neoadjuvant chemotherapy in treatment of osteosarcoma has shown better results, overall survival of patients with metastasis still remains in low rate in the last three decades , 4. Immuγδ T cells, are found favorable for osteosarcoma tumors γδ T cellu et al.  reports u et al.  demonstru et al.  and Tangu et al.  imply thu et al.  and 26]γδ T cellu et al.  that abuγδ T cells and Mast cells. A high infiltration of mast cells has been associated with poor prognosis, low survival and increased metastasis in many cancers [γδ T cells show dual effects on cancer growth [γδ T cells promote tumor development by supporting angiogenesis through angiogenic factors production [γδ T cells have been reported to secrete TGF-β [The observable difference in outcomes between clusters are likely due to the relationship between immune infiltrates and prognosis in osteosarcoma, because we cluster the patients based on their immune composition. Our results indicate that cluster 2 has the worst outcome, while cluster 3 seems to have the best outcome among clusters. The main difference in immune composition between these two clusters is that cluster 2 has much higher percentage of M0 Macrophages, and lower percentage of CD8 T cells and M1 Macrophages than cluster 3. In general, we found that high levels of CD8 T cells and M1 Macrophages are associated with good prognosis, while a high level of M0 Macrophages correlates with poor prognosis in osteosarcoma. These results make sense because CD8 T cells are known to kill cancer cells directly , 48 and  cancers , while γr growth . Both maoduction , 51. Masoduction . γδ T cete TGF-β –54. OverOn the other hand, we did not observe any difference in age or metastasis at diagnosis between clusters. This can be explained by the lack of correlation between immune infiltrates and these clinical variables, which suggests that the immune composition of the primary tumor has no effect on age or metastasis status at diagnosis.Immune checkpoints have an important role in the immune system to prevent autoimmune diseases, but unfortunately they can allow immune tolerance against tumors. PD-1 and CTLA-4 are the main checkpoints that tumor cells use to block immune system –57. Blocγ increases the CD8 T cells expansion [It has been reported that osteosarcoma patients treated with an anti PD-1 drug, Pembrolizumab, show some improvement in disease progression . Combinaxpansion . Thus, pα, IFN-γ, and IL1-β that might be involved in inducing tumor regression [α, IFN-γ, and IL1-β  is another alternative treatment method for osteosarcoma tumors and treatments that suppress M2 Macrophages phenotype or block the polarization of M1 Macrophages to M2 Macrophages have shown positive results in several studies –71. Thus


It renders up to seven dipyridyl polysulfides  which show fully reversible electrochemical behavior in lithium batteries. In the discharge, the initial lithiation occurs at 2.45 V leading to the breakage of Sα—Sβ bonds in Py2Sx and formation of lithium 2‐pyridinethiolate, in which lithium is coordinated in between N and S atoms. The left sulfur species act as elemental sulfur, showing two voltage plateaus at 2.3 and 2.1 V. The molecular dynamics simulations show the attraction between pyridyl groups and lithium polysulfides/sulfide via N···Li···S bonds, which enable good retention of soluble discharge products within electrodes and stable cycling performance. In the recharge, low‐order Py2Sx  remain as the charged products. The mixture catholyte exhibits superlong cycle life at 1C rate with 1200 cycles and 70.5% capacity retention.Organic compounds with active sites for lithiation can be used as electrode materials for lithium batteries. Their tunable structures allow a variety of materials to be made and investigated. Herein, a spectrum of dipyridyl polysulfides  is prepared in electrolyte by a one‐pot synthesis method, which exhibits superlong cycle life of 1200 cycles with 70.5% capacity retention. Its redox reaction mechanism in lithium batteries is revealed.A spectrum of Py The target compound is dipyridyl trisulfide (Py2S3) with a theoretical specific capacity of 425.4 mAh g−1, each Py2S3 molecule could take 4 Li+ and 4 e− when electrochemically reduced in lithium batteries. A mixture of dipyridyl polysulfides (Py2Sx) dissolved in the electrolyte was obtained , dipyridyl pentasulfide (Py2S5), dipyridyl hexasulfide (Py2S6), dipyridyl heptasulfide (Py2S7), and dipyridyl octasulfide (Py2S8) are presented, respectively, indicating the maximum x for a stable dipyridyl polysulfide is 8. The e peak is attributed to the electrolyte. The corresponding m/z values are shown in Figures S1 and S2 in the Supporting Information. In addition, Table S1 in the Supporting Information depicts the proportion of their contents in the ionization mode. Except the unreacted Py2S2, the yield of polysulfides reaches ≈78%. The obtained catholyte was added in carbon nanotube (CNT) paper to be used as cathode.2Sx within the electrode.Herein, Py2S2 to that of Py2S8  to −1.09 eV (Py2S7), although Py2S8 displays a bit higher energy level of −0.97 eV. Based on frontier molecular orbital theory, it is generally considered that the electrons are first injected into the LUMO orbitals during the lithiation process, thus making the high order Py2Sx with low LUMO energies  lithiated first in the discharge of lithium batteries.The electronic structure is vital factor affecting the electrochemical behavior of organic electrodes.8 Figurea, the lo2Sx in batteries is evaluated in lithium half cells, which were discharged and charged galvanostatically. The initial discharge and recharge voltage profiles at C/10 rate are shown in Figure −1, which is about 92.1% of its theoretical capacity. The high discharge voltage plateau occupies almost 50% of the capacity, which is consistent with the 2 e− transfer to form two lithium 2‐pyridinethiolates per molecule. If the average molecular structure is considered as Py2S3, the middle sulfur atom takes the other 2 e− to form one lithium sulfide (Li2S). The corresponding cycling performance of this cell is shown in Figure S4 in the Supporting Information. Over 100 cycles, the capacity is attenuated to 314.6 mAh g−1 which is 80% of the initial discharge capacity. The Coulombic efficiency is over 99.5%.The electrochemical behavior of Py2S2 with two lithium atoms. This may be due to the formation of a simple coordination structure of N···Li···S in the discharged product, in which the overall charge is neutral and the S—S bond may not break. A trace amount of unreacted Py2S2 A still exists. Figure S5 in the Supporting Information gives the corresponding m/z values of three discharge products a, b, and A. The TIC of the recharged product shows the reformation of various Py2Sx  and the other high‐order Py2Sx has depleted. Meanwhile, in the Fourier transform infrared (FTIR) spectra , the stretching band of S–S linkage generally appears in the range of 520–400 cm−1. Py2S2 shows two bands at 426 and 428 cm−1 and Py2Sx shows weak stretching band in this range. After discharge, almost no band of S–S linkage can be seen. Upon recharging, the reappearance of the band at 424 cm−1 illustrates the reformation of S—S bonds. The FTIR results are consistent with those of UPLC‐QTof‐MS.The cycled products were examined by UPLC‐QTof‐MS. The TIC of the discharged product Figure c shows p2Sx, the GFN‐xTB Hamiltonian was used to generate different conformers and rotamers,2S was performed with the semiempirical density functional theory (DFT) method, which includes the D3 dispersion correction accounting for the London dispersion energy.Figure2S.2Sx, the blue surface shown in the electrostatic potential (ESP) energy maps is the most favorable region for electrophilic attack, which mainly arises near N and S atoms. After lithiation, Li atom primarily localizes between N and S sites and forms the stable state of “like complexes.” Indeed, the electron localization function (ELF) analysis further provides a clear signification of that the electron density around N and S atoms shrinks slightly due to the animation of Li atom. Considering there is no valence bond formed, the weak interaction between Li and N/S is easily interrupted by the external electronic field. Thus, at the moment of delithiation, the unperturbed electron density distribution is regenerated, and then the radicals of PyS• reform and are ready for the next cycle of charge.To further reveal the discharged products of Py2Sx can be illustrated in Figure 2Sx, followed by the Sα—Sβ bond cleavage. Due to the strong electrostatic attraction by the N atoms of the pyridyl groups, weak coordination bonds between N and Li are formed . The m/z value of the protonated 2‐pyridinethiol is obtained through the UPLC‐QTof‐MS analysis of the discharge product .Subsequently, the cycling performance of the Li/Py2Sx in Li–S electrolyte has been synthesized from Py2S2 and sulfur as a catholyte for lithium batteries. UPLC‐QTof‐MS analysis confirms the variety of discharged and recharged products. The lithium cell provides a favorable environment for the electrochemical synthesis of Py2Sx in the charge process. Molecular dynamics simulations and UPLC‐QTof‐MS results reveal the dynamic network coordinated by the N···Li···S bonds in the discharged products. The mixture catholyte exhibits 1200 cycles with 70.5% capacity retention at 1C rate. This study reveals the intriguing redox reactions of Py2Sx and provides guidance for the development of high‐capacity and long‐cycle‐life organic cathode materials for lithium batteries.In summary, a spectrum of PyThe authors declare no conflict of interest.Supporting InformationClick here for additional data file.


By a combination of thermal decomposition and dissolution steps, spent NCM could be converted into Li2CO3 and a transition metal oxalate blend, which served as precursors for new NCM. Importantly, it was also possible to individually separate each transition metal during the recycling process, thereby extending the utility of this method to a wide variety of NCM compositions. Each intermediate in the process was investigated by scanning electron microscopy and X‐ray diffraction. Additionally, the elemental composition of the recycled NCM‐811 was confirmed using inductively coupled plasma optical emission spectroscopy and energy‐dispersive X‐ray spectroscopy. The electrochemical performance of the recycled NCM‐811 exhibited up to 80 % of the initial capacity of pristine NCM‐811. The method presented herein serves as an efficient and environmentally benign alternative to existing recycling methods for lithium‐ion battery electrode materials.The demand for lithium‐ion batteries has risen dramatically over the years. Unfortunately, many of the essential component materials, such as cobalt and lithium, are both costly and of limited abundance. For this reason, the recycling of lithium‐ion battery electrodes is crucial to ensuring the availability of such resources and protecting the environment. Herein, a simple and scalable recycling process was developed for the prototypical cathode active material Li Recycle and re‐synthesize: Spent LiNi0.8Co0.1Mn0.1O2 (NCM‐811) from Li‐ion batteries is recovered by a facile and inexpensive recycling approach. After a series of decomposition and dissolution reactions, Ni/Co/MnC2O4 and Li2CO3 are formed, which serve as precursors for the re‐synthesis of NCM‐811. Additionally, the individual separation of the transition metal compounds is possible. In particular, the cathode active material LiNi0.8Co0.1Mn0.1O2 possesses a large theoretical capacity of over 270 mAh g−1, along with a high mean working voltage of approximately 3.8 V vs. Li+/Li.Lithium‐ion batteries are the most pervasive energy storage technology in recent times. In addition to the extensive employment of lithium‐ion batteries in many small‐scale commercial electronics, the development of electric vehicles has bolstered the demand within the automotive industry as well. Notably, the exceptional energy and power densities exhibited by these battery systems arise from the high‐performance electrode materials, for example, graphite at the anode and LiNi2CO3 followed by a sintering step, a mechanochemical regeneration was achieved.2O2 to simultaneously dissolve the transition metals within LiNi1/3Co1/3Mn1/3O2 and generate a chelating agent, which could be subsequently used as a precursor for resynthesizing NCM.N‐methylpyrrolidone (NMP) prior to immersing the active material in citric acid, which raises concerns for large‐scale production due to the toxicity of the employed solvent. It should also be noted that, beyond citric acid, a wide range of alternative acids can be used for the active material dissolution, such as HNO3,. The thermal decomposition in an oxygen atmosphere should also remove electrolyte decomposition products, however, which were absent in our starting material. During cycling, a cathode electrolyte interphase (CEI) is always formed, which consists of organic as well as inorganic products. We assume that the binder material and its decomposition products , which are formed during the heating step, simulate similar conditions as in cycled NCM.Given that current separation techniques enable the thorough separation of casing parts from the active material and current collectors,28, 292 by lithium compounds has been reported as a potential sequestration process.[2 in the form of Li2CO3. By using waste CO2 from industrial processes, the carbon footprint of the recycling process might be reduced even further. The Li2CO3 and the transition metal oxide were easily separated through the addition of water to dissolve Li2CO3. Afterwards, the solid solution of transition metal oxides was extracted from the aqueous Li2CO3 supernatant by centrifugation, while the Li2CO3 was isolated by evaporation. However, a minor LiF impurity fraction was detected, stemming from the decomposition of PVDF. The yield of lithium after the thermal decomposition of NCM‐811 within a CO2 atmosphere is approximately (75.2±0.1) %. Given that fluorides have been shown to negatively influence the structural properties of NCM, it is necessary to remove any fluoride contaminants from the precursors. Consequently, the Li2CO3 is first dissolved in H2O and mixed with a saturated aqueous solution of Ca(OH)2. Li2CO3 and LiF are converted into LiOH and insoluble CaF2, which can be easily separated. After the solvent evaporation, the obtained LiOH is transformed back into Li2CO3 by heating to 600 °C within a flowing CO2 atmosphere. As indicated by the Rietveld refinement in Figure 2CO3 is obtained.2CO3 powder, the cubic morphology of the crystals and the accompanying elemental analysis further corroborate the purity of the separated phase . Moreover, essentially zero transition metal content was detected in the Li2CO3 by ICP and EDX . Furthermore, a precise determination of the carbon content by the Dumas method revealed a value of approximately (15.9±0.1) wt %, which is in good agreement with the theoretical value of 16.3 wt % (Table S2).Research on binding CO process.30, 312CO3 is shown in Figure Fm3‾m). All structural parameters extracted from the Rietveld refinement are provided in the Supporting Information, Table S3. The purity of the transition metal oxide phase was additionally corroborated by the Dumas method, which indicated that essentially no residual carbon from the binder or the carbon additives remain (Table S2). Moreover, EDX and ICP data show that the nominal ratio of the transition metals is maintained . The yield of the transition metal oxide after decomposition is (96.6±0.1) %.The diffraction pattern of the NCM‐811 decomposition product following the separation of the LiIn the next step, the previously obtained oxide is converted into transition metal oxalates through the dissolution of the oxide in 12 M HCl, followed by the addition of 15 mL of 1 M oxalic acid. Once the transition metal oxalates are in solution, a rapid increase of the pH to a value of 4 leads to the precipitation of an oxalate blend in the same stoichiometry as the starting material, which is confirmed by ICP and EDX . Given that the oxalates tend to be hygroscopic, it is likely that the dihydrates are formed, which was confirmed by the Dumas method. The measured weight fraction of carbon is (12.8±0.1) wt %, which is closer to the theoretical value of the dihydrate than to the anhydrous oxalate (Table S2). Based on the transition metal oxalate dihydrate, the yield of the reaction is approximately (88.2±0.1) %. It should be noted that the supernatant exhibited a greenish hue after the extraction of the precipitated oxalates, indicating that the precipitation was not fully complete. However, the addition of excess oxalic acid did not result in further precipitation.0.33Co0.33Mn0.33O2, LiNi0.5Co0.2Mn0.3O2, LiNi0.6Co0.2Mn0.2O2),[2SO4, NiC2O4 can immediately be separated from the solution 2 and MnC4O2, however the black color of the precipitate could arise from the presence of amorphous MnO2.[Given the variety of NCM compositions that exhibit good performance as cathode materials in lithium‐ion batteries ,34, 35n Figure . Through2O4 and MnC2O4 is >85 wt %. However, the major metal fraction for CoC2O4 is approximately 78 wt %. The minority fractions in the transition metal phases represent the remaining Ni/Co/Mn content present in the main fraction. The slightly lower purity of the cobalt fraction arises from the precise control required over the pH for the transition metal separation. In other words, a small amount of nickel remains after the removal of the NiC2O4 fraction and some MnC2O4 is also precipitated upon elevating the pH to fully remove the CoC2O4 fraction. As can be seen in Figure The purity of the individually separated transition metal fractions was determined by EDX Figure . The maj2CO3 can be mixed using the appropriate stoichiometric quantities with a 5 mol % excess of Li (TM/Li=1 : 1.05) and treated at 805 °C in a flowing O2 atmosphere to afford recycled NCM‐811 . The extraction of Li2CO3 using a carbonated water leaching process mildly destabilizes the secondary particles, resulting in an increase in the number of isolated primary particles. In the next step, the formation of the transition metal oxalates leads to a loss of the spherical shape . The average diameter of the particles within the transition metal oxalate blend is approximately 11 μm. After blending the oxalates with Li2CO3 and calcining at 805 °C, the secondary particles are partially sintered, exhibiting fairly irregular shapes with an average diameter of approximately 5 μm .Given that the morphology of NCM depends on the morphology of the precursors, it is important to monitor the morphology of the used intermediates. The evolution of the particle morphology throughout the recycling process, as determined by SEM, is illustrated in Figure S4. The pristine NCM‐811 particles consist of primary particles with an average diameter of approximately 200 nm, which agglomerate to secondary particles with diameters between 10–20 μm. Upon thermal decomposition into the binary oxides, the particles retain a spherical shape; however, the surfaces become coated with Li1.02(Ni0.8Co0.1Mn0.1)0.98O2. However, a slight deficiency of manganese was detected in the recycled NCM‐811, which is likely due to an incomplete precipitation of manganese oxalate during the recycling process. It should also be noted that a low concentration of sodium (2.4 at %) at the particle surface was detected in the XPS spectra, which could stem from the NaOH used to adjust the pH during the oxalate precipitation and could possibly influence the electrochemical performance . While we cannot exclude that a low concentration of sodium ions are also incorporated into the structure, sodium doping of nickel based oxides have been suggested to improve the electrochemical performance of cathode active materials during cycling.[In addition to the morphology, the elemental composition of NCM‐811 also plays a crucial role in the resulting electrochemical performance. High nickel fractions offer high capacities, while increasing the cobalt and manganese content leads to better electronic conductivity and mechanical stability, respectively.45, 46+/Li, whereas for the recycled material the onset is at 3.8 V. Notably, the capacity loss in the first cycle is much larger in the case of the recycled NCM‐811 cell than for the pristine NCM‐811 cell , which suggests that the recycled material possesses a higher surface area. During the first cycle, CEI formation consisting of electrolyte decomposition and cathode surface passivation takes place, which results in some irreversible capacity loss.[The electrochemical performance of the recycled NCM‐811 was investigated with rate capability tests followed by long‐term stability tests using a coin cell architecture. Figure ity loss.51, 52−1 in the first cycle at 0.1 C, which corresponds to 84 % of the capacity of the pristine NCM‐811 electrode. The lower capacities observed in the recycled NCM‐811 cell can most likely be attributed to the previously mentioned undefined particle morphology, given that the morphology strongly influences the intercalation behavior. Well‐defined spherical particles have already been shown to intercalate lithium‐ions more efficiently, thereby achieving higher lithium utilization.Figure 5c shows the rate‐dependent specific discharge capacities over 100 cycles. The recycled NCM‐811 achieves up to 166 mAh gx−y1−CoxMnyO2 (NCM) cathode materials for Li‐ion batteries. The recycling process offers the possibility to reuse the active elements in the cathode material without the need for complex separation processes. First, spent NCM is decomposed into Li2CO3 and Ni0.8Co0.1Mn0.1O. By a simple dissolution process, the oxide is converted into an oxalate, which serves as a precursor for fresh NCM‐811. X‐ray diffraction, inductively coupled plasma optical emission spectroscopy, and energy‐dispersive X‐ray spectroscopy revealed the excellent purity of the Li2CO3, the individually separated transition metal fractions, as well as the recycled NCM‐811. Given that a variety of different NCM compositions are currently being employed in Li‐ion batteries, another crucial factor is the individual separation of the transition metals. This approach offers the possibility to efficiently separate the transition metals, thereby enabling the synthesis of a variety of NCM compositions independent of the starting composition. For the separation of the transition metals components, a simple pH‐dependent precipitation process was used. Furthermore, the electrochemical performance of half‐cells containing the recycled NCM‐811 was investigated and showed an initial specific capacity of 166 mAh g−1 at 0.1 C, which is approximately 84 % of the pristine NCM electrode capacity. Nevertheless, optimization of the NCM‐811 particle morphology could be performed to further enhance the electrochemical performance. Ultimately, the presented method offers a rapid, facile, and scalable alternative to existing recycling approaches for Li‐ion battery electrode materials.Herein, we have demonstrated a rapid and facile process for the recycling of LiNiRecycling procedure: The recycled NCM‐811 was prepared through the decomposition of Li1.02(Ni0.8Co0.1Mn0.1)0.98O2 (BASF SE). First, fresh electrodes of NCM‐811 were prepared by mixing 90 wt % NCM with 5 wt % SuperPLi  and 5 wt % Solef PVDF 5130 (BASF SE). The electrode slurry was stirred for 12 h to ensure a homogenous distribution of all components. Afterwards, a 300 μm thick film was cast on aluminum foil using a doctor blade. The electrode sheet was dried for 24 h in vacuum and subsequently pressed with a roll press  to a thickness of 60 μm. All described steps were performed under an inert atmosphere or vacuum to prevent the exposure to air and avoid side reaction of NCM with moisture. Current processes enable the separation of cell parts at such a level that only a mixture of active materials, binder and carbon remain for the separation.[2 atmosphere and afterwards the active material was decomposed by heating to 600 °C in a flowing CO2 (500 sccm) atmosphere for 60 h. Reaction times shorter than 60 h resulted in only partial conversion of NCM into Li2CO3 and Ni0.8Co0.1Mn0.1O. After decomposition, 1.37 g of the resulting blend  was dispersed in 150 mL distilled water to separate the Li2CO3 from the transition metal oxides. To increase the solubility of Li2CO3, CO2 was also bubbled in the solution. After centrifuging, a solution containing Li2CO3 and a precipitate of transition metal oxides were obtained (0.97 g). Upon evaporating the water from the Li2CO3 solution, LiF contaminated Li2CO3 was obtained (352.50 mg), which arises from the PVDF binder used in the cathode composite. LiF was removed by dissolving the obtained Li2CO3 in water and mixing the solution with a saturated aqueous Ca(OH)2 solution to convert LiF into CaF2, which can be easily removed from the solution due to the low water solubility. Since Li2CO3 is also partly transformed into LiOH during this step, the Li2CO3/LiOH mixture was again heated to 600 °C in a CO2 flow to convert the product back to Li2CO3. From here, the transition metal oxalates were synthesized to be used as precursors for the subsequent NCM synthesis. Therefore, 0.97 g of the transition metal oxide were dissolved in 50 mL concentrated HCl (HCl+10 vol % H2O2) and 15 mL of 1 m oxalic acid was added to the solution. In order to precipitate the oxalates, the pH was increased through the addition of NaOH to the solution. By rapidly increasing the pH to 4, a blend of NiC2O4, CoC2O4, and MnC2O4 was obtained. After the precipitation was finished, the oxalate blend was washed three times with water and dried at 80 °C in vacuum for 12 h. Finally, stoichiometric amounts of the transition metal oxalate and Li2CO3 (5 mol % of excess of Li were used to compensate the Li evaporation during synthesis) were mixed and heated to 805 °C in a flowing O2 atmosphere (200 sccm) to prepare recycled NCM. After the synthesis, the recycled NCM was directly transferred into an argon‐filled glovebox.2SO4 . At this point, NiC2O4 already precipitated and was separated from the solution by centrifugation. After the separation of NiC2O4, the pH was increased through the addition of NaOH. At a pH of approximately 1.5, CoC2O4 precipitated, which was also separated by centrifugation. Finally, by further increasing the pH to nearly 14, a mixture of several manganese species 2) precipitate. All precipitates were centrifuged and washed twice with distilled water.The separation of the transition metals was achieved as follows: first, the synthesized mixed transition metal oxalate blend was dissolved in 15 mL HXRD: Powder XRD was carried out using an Empyrean diffractometer  with CuKα radiation in Bragg–Brentano θ–θ geometry using a PIXcel3D area detector with 255 measuring channels. XRD data were collected in the range 2θ=10–90°. In order to minimize the effect of X‐ray fluorescence of the transition metals, the PHD upper and lower values were set to 70 and 49, respectively.Rietveld analysis: Rietveld refinements were carried out using the TOPAS software package.1.02(Ni0.8Co0.1Mn0.1)0.98O2 (NCM‐811) was refined using the rhombohedral R3‾mspace group (no. 166) as a starting model. For decomposition products, namely Li2CO3 and Ni0.8Co0.1Mn0.1O, the space group C2/c (No. 15) and Fm3‾m(No. 202) were used, respectively. Fit indicators: Rwp, and goodness‐of‐fit were used to assess the quality of the refined structural models.+ and Ni2+ was also determined through the refinement of the occupancy of Ni on the 3b Wyckoff position of Li.ICP‐OES: ICP measurements were conducted using a Varian 720ES instrument with an axial flare, Echelle‐optics, and a CCD detector. Approximately 10 mg of each sample were dissolved in 10 ml of 2 m HCl. The samples were measured with a power of 1.2 kW and the measurement time was 20 s.SEM/EDX: SEM and EDX measurements were conducted with a “Merlin” instrument from Zeiss. For imaging, the accelerating was 5 kV and the current was 120 pA. For the elemental EDX analysis an X‐Max detector from Oxford Instruments was used. The accelerating voltage was increased to 15 kV and the accelerating current 700 pA to obtain elemental maps with sufficient intensity.Cell assembly and electrochemical testing: Electrodes of recycled NCM were made as described above. After the electrode sheet preparation, circular electrodes with 12 mm in diameter were punched out. The cells were assembled as CR2032 type coin cells (MTI Corporation) in an Ar‐filled glovebox (Glovebox Systemtechnik). The cells were tested in a half‐cell configuration using metallic lithium (Rockwood Lithium GmbH) as the anode material and Whatman® GF/A Glass microfiber filters as the separator. 100 μL LP50 (Sigma Aldrich) were used as the electrolyte, which is a mixture of LiPF6 dissolved in ethylene carbonate and ethyl methyl carbonate (50 : 50 w/w). The prepared cells were sealed with a crimper (MTI). The rate capability and the long‐term stability was measured for each cell. Therefore, the cycling protocol contains an initial C‐rate test  and a long‐term test (0.5 C for 90 cycles) was also performed.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Commercially available trifluoroborate UCB‐J analogue was used as a radiolabelling precursor, and the desired radiolabelled product was isolated in 11 ± 2% (n = 7) nondecay corrected radiochemical yield and formulated as a 10% EtOH solution in saline with molar activities of 20 to 100 GBq/μmol. The method was based upon the palladium(0)‐mediated Suzuki cross‐coupling reaction and [11C]CH3I as a radiolabelling synthon. The isolated product was cGMP compliant as demonstrated by the results of quality control analysis.An automated radiosynthesis of carbon‐11 positron emission tomography radiotracer [ PETpositron emission tomographySV2Asynaptic vesicle glycoprotein 2ALCMSliquid chromatography mass spectrometryHPLChigh‐pressure liquid chromatographyTLCthin‐layer chromatography1Synapses are specialized structures in the brain, which mediate a functional interaction between two neurons or between a neuron and another cell type.1; Figure 11C]2; Figure Recently developed is a noninvasive alternative using positron emission tomography (PET), which for the first time enables the quantification of synaptic density in vivo. This is achieved by radiotracers selective to synaptic vesicle glycoprotein SV2A, a transmembrane protein expressed ubiquitously in secretory vesicles in all brain areas.11C]UCB‐J for which a radiosynthesis method has been developed.11C]UCB‐J using Synthra RNPlus automated radiosynthesizer.Therefore, to undertake clinical SV2A PET studies at our centre, our objective was to establish a manufacturing method for CUCB‐J fo22.1All anhydrous solvents were purchased from Aldrich, Alfa Aesar, or Acros and used as received unless otherwise noted. Water for injection was purchased from Braun and sodium chloride 0.9% w/v injection BP from Kent Pharmaceuticals. Water for cleaning of the synthesizer was Millipore (18.2 MΩ), and water used for the radiosynthesis was BP grade. The radiolabelling precursor and the reference material were obtained from UCB, Belgium. The Sep‐Pak Classic C18 cartridges were purchased from Waters and the empty 3‐mL SPE columns with frits Chromabond from Macherey‐Nagel. Ethanol for injection was purchased from Merck.11C]CH3I and decay corrected unless otherwise stated.Radiochemical yields for the product were based on [2.22O and absolute EtOH and acetone. The [11C]CH3I delivery line was tested for flow and leak tightness. The GE MeI module was preconditioned (heating followed by the cooling of the Ni‐ and porapak ovens) using automated PREPARATION stage of the module.Prior to each production, the automated synthesizer was cleaned using HA3: 0.3 mL DMF:H2O 8:1 v/vB3: 1.6 mL 1M aq HClC1: 13 mL NaCl 0.9% BPC2: 1.4 mL absolute EtOHC3: 15 mL H2O for injectionSPE: 50 mL H2O for injectionC18: Classic cartridge, conditioned with 5 mL EtOH, 10 mL H2O, then 2 mL airFilter frit: filled with 3.2 mL HPLC eluent, placed between V47 and V25HPLC: Semipreparative column Gemini‐NX C18 250x10 mm, 5 μm, 100 Å, eluting with 65% 100 mM ammonium formate pH 9.2 and 35% MeCN; flow 8 mL/min, λ = 254 nmREACTOR 1: 1.6‐1.7 mg (R)‐trifluoropyrrolidin‐1‐yl)methyl)pyridin‐1‐ium‐3‐yl)borate (UCB‐J precursor) in 54 μL MeOH and 22 μL 1M aq HClREACTOR 2: 28 μL potassium carbonate aqueous solution (4‐5 mg in 280 μL H2O), 244 μL bis (dibenzylideneacetone)palladium(0) solution in anhydrous DMF (1.0‐1.5 mg Pd (dba)2 in 488 μL DMF), 200 μL tris(o‐tolyl)phosphine solution in anhydrous DMF (2.0‐2.5 mg P(o‐Tol)3 in 500 μL DMF), and 500 μL anhydrous DMFPRECURSOR ACTIVATION: Precursor activation is a step that commences at the same time as the cyclotron target bombardment starts and precedes the oxidative insertion of Pd to carbon‐halide bond. The activation of the precursor is achieved by placing the precursor into the reactor 1, adding MeOH and HCl, and stirring the mixture over 30 minutes at ambient temperature. The mixture is then dried in the stream of He and under vacuum and finally only vacuum after which it is redissolved in DMF:H2O  and transferred from reactor 1 to reactor 2.The automated Synthra RNPlus set up was as follows 11C using GE PETtrace cyclotron from 0.5% O2 enriched N2. Typically bombardment was performed at 35 μAh over 40 to 45 minutes. The produced [11C]CO2 was then converted to [11C]CH3I using a GE MeI module via reduction to [11C]CH4 using a Shimalite nickel catalyst/column followed by condensation reaction of [11C]CH4 with I2 at 720°C during the recirculation after which [11C]CH3I is trapped on the Porapak and released upon heating. The produced [11C]CH3I was received in the reactor 2 of the Synthra RNPlus automated module and bubbled through the mixture containing base, ligand, and catalyst solution and DMF (see REACTOR 2) at –15°C for 4 to 7 minutes during the number of counts per second in the reactor 2 peaked. The reaction mixture was then heated to 30°C for 3 minutes to allow for oxidative insertion of Pd to carbon‐halogen bond to occur. The activated precursor was then added (see PRECURSOR ACTIVATION) as DMF:H2O solution, and the reaction mixture was allowed to heat to 100°C over 5 minutes. After this time, the reaction mixture is allowed to cool to 30°C and then quenched with HCl from B3 and loaded onto the high‐pressure liquid chromatography (HPLC) loop via the syringe and by passing the mixture through the SPE column with frit containing eluent. The semipreparative purification finally yields desired [11C]UCB‐J, which is collected in the SPE flask containing H2O and then trapped on the C18 Classic cartridge. The product is washed with H2O and then eluted with EtOH and formulated as a saline solution and dispensed in the grade A isolator by passing the mixture through the sterile filter.[2.311C]UCB‐J took approximately 30 minutes and was performed as described previously.11C]UCB‐J peak on the radio‐chromatogram. Radiochemical identity was confirmed and assessed by comparison of the retention time of the [11C]UCB‐J radioactive peak to that of the UCB‐J reference standard peak in the coinjection sample. The chemical amount of [11C]UCB‐J and cold impurities was determined by quantification and relative comparison of the corresponding UV absorbance peaks of the QC sample and reference standards of known concentration. The acceptance limit of 10 μg for UCB‐J in the injection dose was established based on the selected animal toxicity study taking into account guideline on setting permitted daily exposure by European Medical Agency. The maximal allowable injection volume (Vmax) of radiotracers produced at the Wolfson Brain Imaging Centre (WBIC) is by default limited to 10 mL and was calculated based on the chemical amount of [11C]UCB‐J and cold impurities relative to their specification Vmax = 10 μg/specification of [11C]UCB‐J found in a QC sample (μg/mL). Molar activity (Am) was calculated as the ratio of radioactivity in GBq and the amount of UCB‐J (μmol) at the time of anticipated application (20 min after the EOS). The integrity of the Acrodisc Supor Membrane filter  was tested using the filter integrity  test. Sterility, bacterial endotoxins, radionuclidic identity, and residual solvent analyses of the product were assessed as the postrelease tests. Endotoxin content in [11C]UCB‐J doses was measured with the Charles River Laboratories Endosafe Portable Test System. Residual solvent analysis was performed on Thermo Focus Gas Chromatograph equipped with the Thermo column (6′×1.8′×0.85″) and a flame ionization detector. Radionuclidic identity was determined by measuring [11C]UCB‐J activities in triplicate at different time points  using Veenstra VDC‐505 radioisotope dose calibrator, and the half‐life was calculated as per the following: t1/2 = –ln2·((t2 – t1)/ln(A2/A1)), whereby t1 and t2 are two different times and A1 and A2 are radioactivity measured at times t1 and t2, respectively.Prerelease QC of [311C]UCB‐J is based on the application of the palladium(0)‐mediated Suzuki cross‐coupling reaction between the carbon‐11 methyliodide ([11C]CH3I) and trifluoroborate precursor 3 . In methanolic HCl, a 24% conversion by LCMS was achieved after 40 minutes (entry 2). Using the aqueous solution of HCl (c=1 M) afforded desired 4 in maximum 39% LCMS conversion after repeated addition of the acid and extended reaction time. We speculated that it was the concentration of the reaction mixture, which determined the conversion to the desired boronic acid 4. To test this, the aqueous HCl used by Onega et al3 and boronic acid 4 was required to be free from MeOH/HCl prior to Suzuki cross‐coupling reaction. The smaller volume of solvents (MeOH/HCl) would be quicker to remove; thus, we set out to use 63 mM concentration and perform the radiochemical step next. To implement an automated method for [11C]UCB‐J manufacture, we decided to use a Synthra RNPlus module  two reactors that are ideal for the in situ preparation of the reacting precursor 3/4 and subsequent Pd‐mediated Suzuki coupling; (b) size of the reactors which allows for larger volumes particularly for the injection of crude reaction mixture onto semipreparative HPLC column; spare valves that allow for introduction of external gases, eg, [11C]CH3I; (d) spare connection points for the filter frit and the SPE cartridge to allow filtration of the crude mixture and product formulation; and (e) easily replaceable HPLC injection loop and corresponding injection syringe to allow for 5‐mL injection volumes.While the main disadvantage of using Synthra RNPlus module, which is intended for radioactive fluorinations, was a requirement for the external source of CH3I (I to II), ie, oxidative addition, must precede transmetallation (III to IV) for which reason the activated precursor 3/4 is added once the [11C]CH3I trapping is complete and reactants allowed time for oxidative insertion. Allowing the oxidative insertion step to occur ahead of the addition of boron analogue enabled the successful conversion to the product as has been demonstrated previously for other Suzuki transformations.The availability of two reactors proved beneficial for two reasons. It was quickly determined that the activation/hydrolysis of precursor n Scheme , Pd‐comp11C]CH3I was converted to [11C]UCB‐J as assessed by radio‐HPLC using the following setup: reactor 1 was charged with the precursor 3/4, MeOH and 1M aq HCl; reactor 2 was charged with Pd(0)‐catalyst, (o‐Tol)3P and the base K2CO3 . The reliable radiosynthesis was achieved however, only if the reactor was pressurized immediately after the [11C]CH3I trapping, significantly limiting the radioactivity losses  and if decay corrected 35 ± 4% (n = 7). The QC analysis of the produced [11C]UCB‐J indicated that the product conforms to cGMP conditions as demonstrated by the summarized QC data in Table Following closely these requirements, conversions of up to 90% were reproducibly achieved. The radiosynthesis was successfully programmed, and typical automated profile is depicted in Figure 411C]UCB‐J using the Synthra RNPlus automated synthesizer has been achieved with RCY at end of synthesis comparable to that reported and in compliance with the cGMP regulations. Several modifications to both the reaction conditions  and the Synthra RNPlus module  were required to allow for a reliable production protocol and led to identification of critical reaction parameters. This in turn allows for the implementation of the described production protocol in other clinical PET centres for routine manufacturing of [11C]UCB‐J.In summary, reproducible radiosynthesis of UCB‐J using the Synthra RNPlus platform is described.An automated radiosynthesis of [11C]UCB‐J on an automated synthesizer Synthra RNPlus has been described starting from the trifluoroborate precursor and using carbon‐11 labelled CH3I as radiolabelling synthon.The radiosynthesis of [Table S1. Amounts and volumes of UCB‐J precursor 3 and HCl/MeOH for hydrolysis under unlabeled conditionsClick here for additional data file.


Solanum tuberosum group Andigena (Solanum andigena), short days, or actually long nights, induce tuberization. Applying a night break in the middle of this long night represses tuberization. However, it is not yet understood how this repression takes place. We suggest a coincidence model, similar to the model explaining photoperiodic flowering in Arabidopsis. We hypothesize that potato CONSTANS (StCOL1), expressed in the night of a short day, is stabilized by the light of the night break. This allows for StCOL1 to repress tuberization through induction of StSP5G, which represses the tuberization signal StSP6A. We grew S. andigena plants in short days, with night breaks applied at different time points during the dark period, either coinciding with StCOL1 expression or not. StCOL1 protein presence, StCOL1 expression and expression of downstream targets StSP5G and StSP6A were measured during a 24‐h time course. Our results show that a night break applied during peak StCOL1 expression is unable to delay tuberization, while coincidence with low or no StCOL1 expression leads to severely repressed tuberization. These results imply that coincidence between StCOL1 expression and light does not explain why a night break represses tuberization in short days. Furthermore, stable StCOL1 did not always induce StSP5G, and upregulated StSP5G did not always lead to fully repressed StSP6A. Our findings suggest there is a yet unknown level of control between StCOL1, StSP5G and StSP6A expression, which determines whether a plant tuberizes.In the obligate short‐day potato  HAhemagglutininLDlong dayNBnight breakPHYBphytochrome BRTroom temperatureSDshort daySDSsodium dodecyl sulfateStCOL1StCONSTANS-like 1ZTZeitgeber timeSolanum tuberosum) is the third most consumed food crop by humans (http://cipotato.org/potato). Gaining an extensive understanding on tuberization (tuber formation) can give growers the tools that are needed to optimize growth and tuber yield.Globally, potato (Solanum tuberosum group Andigena (Solanum andigena) is a variety that originated in the Andes, and is used as a model potato plant for photoperiod studies, because of its strict short‐day requirement for tuberization . Orthologs of these genes are responsible for photoperiodic control of flowering  under long‐day conditions  expression is controlled by circadian clock components, which at the end of a long day form a complex that degrades CYCLING DOF FACTOR (CDF), an inhibitor of CO, allowing for CO expression at the end of a long day. In short days, CDF is not degraded by this complex, only allowing CO to be expressed in the night. Post‐translational control of CO by light further determines whether flowering is induced. In the light, photoreceptors PHYTOCHROME A  and CRYPTOCHROME  reduce the activity of a complex that degrades CO. In short days, CO is only expressed in the night where CO is degraded. In long days, CO expression takes place in the light where CO is not degraded, leading to the induction of FT and the initiation of flowering. A similar coincidence of light and internal gene expression also controls flowering time in the short‐day plant rice. An ortholog of CO in rice called HEADING DATE 1 (Hd1), induces a rice FT, Hd3a, in short days, but represses it in long days  , which represses the FT‐like tuberization factor SELF‐PRUNING 6A  activates PHYB and far‐red light (730 nm) inactivates it Casal . In daylStCOL1 expression in a short day only peaks at the end of the night, and is low in the middle of the night when a night break is generally applied  tag (35S::StCOL1‐HA) in an S. andigena background were used for later protein detection by western blot. The transformation was done as described in Navarro et al.  that were filled with a clay‐peat mixture and placed in two climate chambers. All plants were watered manually and liquid fertilizer was given once per week . The climate chambers were set at 20°C day and night, with a relative humidity of 70%. The plants were illuminated by red and white LEDs . LED height was adjusted every 2 weeks to maintain the required intensity . The light intensity was measured at the top of the plant canopy with a quantum sensor . Plants were rotated three times a week to ensure uniform light distribution and all side‐shoots were removed from the plants.The plants were propagated in vitro on MS20 medium (Murashige and Skoog 35S::StCOL1‐HA line were placed in each light compartment. The light intensity applied was 300 μmol m−2 s−1 (short day), 282 μmol m−2 s−1 (short‐day + night‐break treatments) or 150 μmol m−2 s−1 (long day) . Four weeks after transplanting, plants were sampled for gene expression analysis and protein detection at 11 time points during a 24‐h cycle  SD: a short‐day control treatment with 8/16 h light/dark, 2) NB12: a short day with a 30‐min night break early in the dark period (ZT(12); Zeitgeber time, hours after the start of light period), (3) NB16: a short day with a 30‐min night break in the middle of the night (ZT(16)), (4) NB20: a short day with a 30‐min night break towards the end of the dark period (ZT(20)) and (5) LD: a long‐day control treatment with 16/8 h light/dark. The night breaks were given every night, from the moment the plants were placed in the climate chamber until harvest. The light given during the night breaks was identical to the light given during the day and consisted of red and white LEDs. The treatments were divided over two climate chambers; one climate chamber was divided into four compartments with short‐day treatments and the other climate chamber contained the long‐day control treatment. Thirty‐three plantlets of the wild‐type and 33 plantlets of the  NB12: a ACTIN primers (see below) on DNase‐treated RNA samples (−RT control). The purified RNA was synthesized to cDNA using a high‐capacity cDNA reverse transcription kit . DNase treatment and cDNA synthesis were performed as per the manufacturer's instructions. Twenty microliters of cDNA was diluted with Milli‐Q water to a total volume of 200 μl. The qPCR mix contained 5 μl SYBR‐green , 0.5 μl forward primer (10 μM), 0.5 μl reverse primer (10 μM), 1.5 μl Milli‐Q water and 2.5 μl cDNA. The qPCR was performed with the following program in a Thermal Cycler : 95°C for 3 min, then 40 cycles alternating between 95°C for 10 s and 58°C for 30 s. The qPCR was followed by 95°C for 10 s and a melt curve analysis from 65 to 95°C with 0.5°C increments, each step for 10 s. Three biological replicates were used per time point in each treatment and a negative‐control (no cDNA) was done per primer‐pair. The primers for the target genes StCOL1, StSP5G and StSP6A and the reference genes StEIF3e and StACTIN are given in Fig. Gene expression was determined using reverse transcription quantitative polymerase chain reaction (qPCR). For RNA extraction, the frozen plant material was ground to powder with a ball mill. Approximately 25 mg of plant sample was placed in a tube, to which 1.2 ml of TRIzol reagent was added . After mixing and incubating at room temperature (RT) for 5 min, samples were centrifuged at maximum speed at RT in a table‐top centrifuge. One milliliter of the supernatant was transferred, to which 250 μl chloroform was added and shaken. After centrifugation, 450 μl of the aqueous phase was mixed with 1 volume of isopropanol and samples were incubated at RT for 20 min. After centrifugation, the pellet was washed with 70% ethanol and dissolved in Milli‐Q water. Several RNA quality steps were performed Taylor ; the intProtein detection was done using western blot analysis. Hundred microliters of extraction buffer  was added to approximately 50–100 μg of ground frozen material. After thawing, the samples where vortexed and placed at RT for 5 min after which they were centrifuged at 4°C for 10 min. The supernatant was collected and loaded onto a 10‐well gel . Protein separation was done with SDS–polyacrylamide gel electrophoresis (SDS–PAGE) in a PowerPac  with a running buffer  at 100 V and was run until the dye ran out of the gel (approximately 110 min). The gel was then blotted onto a methanol‐activated membrane . The sandwich containing gel, membrane and filter paper sponges was placed in a transfer buffer  and was run for 90 min at 300 mA in the PowerPac. After blotting, the membrane was rinsed in demi water and stained with a Ponceau solution  to obtain a loading control. After staining, the membrane was rinsed in Tris‐buffered saline  and soaked in a milk‐TBS mix (5% milk powder Elk Campina in TBS) for 1 h. The membrane was then placed in a falcon tube containing 5 ml of 5% milk TBS buffer to which 5 μl of anti‐HA antibody was added  and incubated overnight at 4°C. After incubation, the membrane was washed three times in TBS‐T  and one time for 5 min in TBS. Protein presence was detected using 0.5 ml SuperSignal West Femto Chemiluminescent substrate (Thermofisher) and detected in a chemiluminescence imager .anova and Fisher's protected lsd (α = 0.05) for pairwise comparisons. These analyses were computed in genstat (18th Edition). Due to confined space in the climate chamber, there were no repetitions of light treatments and only biological replicates were present. Therefore, every plant was considered as an independent experimental unit. The qPCR was performed with three biological replicates per time point and treatment, with the exception of 35S::StCOL1‐HA samples, which were pooled samples of three plants and had three technical replicates instead. qPCR data were obtained from cfx manager (Bio‐Rad) version 3.1 and expression levels were calculated with 2−ΔCt  and the geometric mean of the two reference genes (EIF3e and ACTIN). For the western blot analysis, one sample was used per time point and treatment. The sample was a pool of leaflets from three plants.The tuberization and flowering times between treatments were compared with a one‐way S. andigena was grown under 8 h of light and 16 h of darkness, tuberization occurred after 28 days , the time the night break was applied. In the treatment with the night break given toward the end of the dark period (NB20), the light of the night break coincided with peak expression of StCOL1  did not coincide with the light of the night break, but StCOL1 expression in the early night‐break treatment was high in the light period at ZT(2), which was not the case in the other treatments. StSP5G was induced in the early night‐break treatment , and StSP5G was highly expressed compared to the other treatments . By using an overexpressing line, we were able to observe when the StCOL1 protein was absent and thus determine when it was degraded. By comparing this information with the StCOL1 expression pattern in wild‐type lines, it could be determined when StCOL1 protein could be present in the leaves. Under short‐day conditions in the overexpressing line, StCOL1 was present in the light and degraded in the dark period  occurred more than 2 weeks later than in the wild‐type plants, while tuberization in the early night‐break treatment (NB12) did not occur at all when StCOL1 was overexpressed ) and peak StCOL1 expression (ZT(20); Fig. StCOL1 expression to repress tuberization. What is even more contradictory to the hypothesis that the coincidence between light and StCOL1 expression represses tuberization are the unexpected results of the late night‐break treatment NB20. In this treatment, the coincidence of light and peak StCOL1 expression at ZT(20) did not lead to high StSP5G expression or repressed tuberization  tuberized after approximately 32 days, while StCOL1 peak expression coincided with light only 4 days earlier.An alternative explanation is that light activates PHYB, which is only slowly deactivated by dark reversion  in the NB20 treatment indicates that there is an extra level of control between StCOL1 and StSP5G that still needs elucidation or that StSP5G is regulated by a transcription factor other than StCOL1. Furthermore, the upregulation of StSP6A despite upregulated StSP5G in the NB12 treatment suggests that StSP5G expression alone does not determine StSP6A induction.Light‐stabilized StCOL1 during the night cannot explain the repressive function of a night break. The lack of StCOL1 and light did not always lead to strong repression of StSP6A expression and of tuberization. This lack of correlation was also seen in the plants overexpressing StCOL1. Although StCOL1 was expressed throughout the day in both short‐ and long‐day conditions, and StSP5G expressed in both day‐lengths, StSP6A was only induced in short days. This confirms our earlier finding that upregulation of StSP5G does not necessarily lead to repression of StSP6A. StSP5G is not a transcription factor and needs an additional factor to affect StSP6A expression. Abelenda et al.  had a short dark period followed by a night break and a longer dark period. Instead of the length of the dark periods, the time until the first light period may be important for successful repression of tuberization.Before anything was known about the molecular control behind tuberization, a night break was claimed to repress tuberization by dividing a long night into two short nights Jackson . Our resPPD‐B1, an allelic variant of PHOTOPERIOD1 (PPD1) whose expression leads to FLOWERING LOCUS T1 (FT1) induction in wheat and may be comparable to StCOL1 in potato. However, the increase in PPD‐B1 did not correlate to flowering time, suggesting that other circadian clock genes also play a role in night‐break induced flowering in wheat. Interestingly, more parallels exist between night breaks in wheat and potato. Although StCOL1 was expressed at a low level during a classic night break, StSP5G was induced and tuberization was repressed. In wheat, the greatest night‐break‐mediated flower induction was observed in the middle of the night when PPD1 expression was low  may coincide with an early low expression of this unknown gene, while during the late night break (NB20) the photosensitive period is over, explaining the low StSP5G expression, even while StCOL1 is stabilized. Although such a mechanism involving an additional factor influenced by the night duration is plausible, not all results can be explained. For instance, why do StCOL1‐overexpressing plants have similar high StSP5G levels if only in long days the additional factor induces StSP5G? Possibly both this factor and StCOL1 act on StSP5G. Alternatively, this additional factor may stabilize or activate StSP5G or even repress StSP6A independently of StSP5G, explaining why StSP6A is fully repressed in long days in StCOL1‐overexpressing lines, while in short days some StSP6A expression can still occur.Pearce et al.  found thStSP5G and StSP6A, complicating the existing StCOL1 controlled model explaining potato tuberization. An additional factor that is controlled by the duration of the dark period may be a missing link in the control of photoperiodic tuberization. Three tandemly arranged orthologs of CO were identified in potato (StCOL1‐StCOL3) and it was shown that StCOL1 is expressed at relatively high levels in leaves, but StCOL2 levels are much lower and StCOL3 is almost undetectable (Abelenda et al. StCOL2 was shown to peak in the middle of a long night, which fits with the expected action period of the additional controlling factor. Additionally, Abelenda et al. (StSP5G expression. Determining the tuberization phenotype and StSP5G and StSP6A expression patterns in transgenic plants silenced in StCOL2 may determine if this paralog of StCOL1 is involved in the regulation of a night break.A complex mechanism may take place where multiple factors act on a et al.  demonstrStCOL1 expression and light cannot explain why a night break represses tuberization in short days. Night‐break treatments that were given during peak expression of StCOL1 did not strongly induce StSP5G nor substantially repress StSP6A expression. Furthermore, plants overexpressing StCOL1 still expressed StSP6A in short days and were able to tuberize, even though StSP5G expression was almost identical to expression in long‐day plants, which did not tuberize. Our findings suggest there is an additional level of control between StCOL1, StSP5G and StSP6A expression, which determines whether a plant tuberizes.Our results show that coincidence between F.D.M.P. was involved in conception and design of study, acquisition of data, analysis and interpretation of data, drafting the article and final approval of the submitted version. E.H. contributed to obtaining of funding, conception of study, critical revision, statistical expertise and final approval of the submitted version. J.A.R. contributed to acquisition of data, interpretation of data, critical revision and final approval of the submitted version. R.G.F.V. contributed to obtaining of funding, critical revision and final approval of the submitted version. C.W.B.B. and L.F.M.M. contributed to obtaining of funding, conception of study, critical revision and final approval of the submitted version.Fig. S1. Primers used in the study and validation of target gene amplification.Fig. S2. Flowering time in Solanum andigena grown in light treatments with or without a night break.Click here for additional data file.