Multiple Myeloma (MM) is a progressive plasma cell neoplasm characterized by heterogeneous clonal expansion. Despite promising response rates achieved with anti-BCMA CAR-T cell therapy, patients may still relapse and there are currently no clear therapeutic options in post-CAR-T settings. In this report, we present a case of a post-BCMA CAR-T relapsed/refractory (RR) MM patient with skin extramedullary disease (EMD) in which a novel MAPK inhibition combinatorial strategy was implemented based on next-generation sequencing and in vitro experiments.A 61-year-old male with penta-refractory MM penta- (IgA lambda), ISS stage 3 with hyperdiploidy, gain of 1q21 and del13 was treated with anti-BCMA CAR-T cell therapy, achieving a best response of VGPR. He progressed after 6 months and was salvaged for a short period with autologous stem cell transplantation. Eventually, he progressed with extramedullary disease manifested as subcutaneous nodules. Based on whole-exome sequencing, we identified a BRAF (V600E) dominant subclone in both bone marrow and cutaneous plasmacytoma. Following in vitro experiments, and according to our previous studies, we implemented a triple MAPK inhibition strategy under which the patient achieved a very good partial response for 110 days, which allowed to bridge him to subsequent clinical trials and eventually achieve a stringent complete response (sCR).Here, we show the applicability, effectiveness, and tolerability the triple MAPK inhibition strategy in the context of post-BCMA CAR-T failure in specific subset of patients. The triple therapy could bridge our hospice bound RRMM patient with BRAF (V600E) to further therapeutic options where sCR was achieved. We will further evaluate triple MAPK inhibition in patients with BRAF V600E in a precision medicine clinical trial launching soon.The online version contains supplementary material available at 10.1186/s13045-022-01330-3. Relapsed/Refractory Multiple Myeloma (RRMM) patients with extramedullary disease (EMD) have limited response to existing strategies like immunomodulatory drugs, proteasome inhibitors, and monoclonal antibodies. While anti-BCMA CAR-T has achieved prominent response in RRMM patients, post-CAR-T relapse represents a challenging therapeutic course with poor prognosis. About 53% of RRMM patients have shown emerging clones harboring mutations within the MAPK signaling pathway, including the targetable BRAF(V600E) in 7% of cases. TargetiA 61-year-old male with penta-refractory MM (IgA lambda), ISS stage 3 with hyperdiploidy, gain of 1q21 and del13 was treated with anti-BCMA CAR-T cell therapy, achieving a best response of VGPR. He progressed after 6 months and was temporarily salvaged with BCNU/Melphalan  as conditioning regimen before ASCT, achieving a best response of PR until further progression with extramedullary disease (subcutaneous skin lesions in lower extremities) and elevated lambda free light chains  at 6 months  mutation in his BM, which persisted throughout treatment and was also detected in a cutaneous plasmacytoma with a variant allele frequency (VAF) of 41% at relapse from CAR-T. Western blot (WB) analysis of the post-CAR-T relapse bone marrow aspirate (BMA) confirmed the BRAF(V600E) mutation and further showed phosphorylation of ERK (pERK), consistent with activation of MAPK signaling, which was inhibited by combining the MEK inhibitor binimetinib with the BRAF(V600E) monomer inhibitor encorafenib. Imaging the blot again with longer exposure time revealed small residual MAPK activity, which was abrogated by adding the BRAF dimer inhibitor regorafenib Fig. A and C. ion Fig. B [6]. Ac00E mutatBased on these findings and assessment, the patient was started on targeted therapy with a combination of a BRAF monomer inhibitor, dabrafenib , a MEK inhibitor, trametinib , and a BRAF dimer inhibitor, regorafenib . Within 3 months of treatment initiation, prompt reduction in subcutaneous skin lesions and 80% reduction in Lambda free light chain (λFLC) (27.5 mg/l) were observed , trametinib 2 mg , and regorafenib 80 mg , for less than a month, and eventually discontinued when no further response was observed with FLC peaking at 116.6 mg/l . Moreover, the patient has shown tolerability to the administered regimen, as the addition of regorafenib allowed lower doses of both dabrafenib and trametinib, the latter being cardiotoxic at higher doses , 11.After three months, the patient response to the triple therapy declined and was evident by gradual rise of the FLC but with no recurrence of the subcutaneous nodules. This indicates that the triple therapy could successfully eradicate the MM cell clones responsible for the cutaneous manifestation. WES analysis of the patient BMA at relapse from triple MAPK inhibition therapy has also shown a newly emerging clone harboring a KRAS Q61R mutation in addition to the previously existing BRAF V600E, which was associated with increased MAPK activity observed at the RNA level. This may explain the gradual decline of the patient response to the triple regimen.+ signaling analysis post-triple MAPK inhibition by WB indicated that the patient might benefit from copanlisib, a PI3K inhibitor, either as a single agent or in combination with triple MAPK inhibition. Copanlisib could efficiently inhibit the phosphorylation levels of AKT and showed clones dependence on PI3K evident by marked drop of phosphorylation of ribosomal S6 protein compared to MAPK inhibition that showed only a marginal drop .In this case study, we have shown the applicability, effectiveness, and tolerability of the triple MAPK inhibition in hematological malignancy context to salvage post-BCMA CAR-T cell therapy in a RRMM patient with V600E mutation. This represents proof-of-concept that, while not curative, targeted molecules may serve as potential bridging therapies to clinical trial enrollment. We have also demonstrated the power of routine NGS and WB signaling pathway analysis in tracking clonal evolution and identifying targetable pathways which allow tailoring of personalized therapeutics for MM patients.+ selected cells were collected from BMA at multiple time points throughout the disease course from before CAR-T cell therapy to after relapse from triple MAPKi therapy. Genomic DNA was extracted from peripheral blood and sequenced as a matched normal control for variant calling. Additionally, a full BMA was collected from a patient’s cutaneous plasmacytoma sample at CAR-T cell therapy relapse for targeted sequencing via the FoundationOne Heme panel.CD138Raw reads were trimmed and assessed for quality control using fastp . TrimmedPI3KCA, IGF1, IGFR, AKT1, VEGF, VEGFR, and PDK1 [ETV1, ETV4, ETV5, FOS, FOSB, FRA1, MYC, DUSP4, DUSP6 [Raw reads were trimmed and assessed for quality control using fastp , followeand PDK1 . The pat4, DUSP6 .Cells were washed with PBS and lysed on ice for 5 min in NP40 buffer  supplemented with protease and phosphatase inhibitors (Roche). Lysates were centrifuged at 15,000 rpm for 10 min, and the protein concentration was quantified using BCA (Pierce). Proteins were separated by NuPAGE and 4% to 12% Bis Tris Gel (Novex), and they were immunoblotted and transferred to nitrocellulose membranes  according to standard protocols. Membranes were immunoblotted overnight with antibodies against pERK1/2Thr202/Tyr204 (D13.14.4E), pAKTSer473 (D9E), pS6 Ribosomal Protein Ser240/244 (D68F8) and β-actin (13E5) from Cell Signaling; BRAFV600E from NewEast Biosciences. The next day, membranes were probed with anti-rabbit IgG or anti-mouse IgG secondary antibody , and chemiluminescent signals were detected on X-ray films.+ cells’ sensitivity to MAPK inhibitors were assessed by Travera. Selected cells are incubated with different inhibitors at different concentrations and combinations for 15 h before having their single-cell mass distributions measured. Inhibitor efficiency is then characterized by the relative statistical difference (Hellinger distance) between each test condition and untreated controls.RRMM patient’s BMA selected CD138Encorafenib, trametinib, copanlisib, binimetinib, regorafenib were obtained from Selleckchem. Compounds were dissolved in DMSO to yield 10 mmol/L stock.Additional file 1. Laboratory measurement levels of light chain values.


This committed step is the sum of multiple ping-pong electron transfers involving multiple substrates, intermediates, and products all sharing the same active site. Insight into folate substrate binding is needed to better understand this multifunctional active site. Here, we performed activity assays with Thermus thermophilus MTHFR (tMTHFR), which showed pH-dependent inhibition by the substrate analog, N5-formyltetrahydrofolate (CHO-H4folate). Our crystal structure of a tMTHFR•CHO-H4folate complex revealed a unique folate-binding mode; tMTHFR subtly rearranges its active site to form a distinct folate-binding environment. Formation of a novel binding pocket for the CHO-H4folate p-aminobenzoic acid moiety directly affects how bent the folate ligand is and its accommodation in the active site. Comparative analysis of the available active (FAD- and folate-bound) MTHFR complex structures reveals that CHO-H4folate is accommodated in the active site in a conformation that would not support hydride transfer, but rather in a conformation that potentially reports on a different step in the reaction mechanism after this committed step, such as CH2-H4folate ring-opening. This active site remodeling provides insights into the functional relevance of the differential folate-binding modes and their potential roles in the catalytic cycle. The conformational flexibility displayed by tMTHFR demonstrates how a shared active site can use a few amino acid residues in lieu of extra domains to accommodate chemically distinct moieties and functionalities.The flavoprotein methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of N5, N10-methylenetetrahydrofolate (CH Indeed, there has been a rash of interest in enzymes critical to one carbon metabolism, given that their disfunction has been implicated in diseased states (2-H4folate) to produce N5-methyltetrahydrofolate (CH3-H4folate) (A). The overall MTHFR reaction follows a ping-pong bi-bi mechanism , MTHFR uses NAD(P)H , the reduced FAD transfers a hydride to CH2-H4folate to produce CH3-H4folate. Menadione acts as an electron acceptor from the reduced FAD in MTHFR and is thus routinely used as an artificial oxidant in in vitro assays to decouple and separately examine both the NAD(P)H- and CH3-H4folate–dependent  FAD reducing half reactions (Thermus thermophilus HB8 (tMTHFR) (versus a tetramer in Escherichia coli (eMTHFR)  . MTHFR r4folate) A. The ovechanism . In the echanism A, top, M NAD(P)H , 8, 9 to NAD(P)H A, bottom NAD(P)H-A, top anependent A, bottomeactions , despite(eMTHFR) . Eukaryoactivity , 11. Thiynthesis , 11, 12.E. coli (T. thermophilus (Haemophilus influenzae (PDB 5UME), human . In the catalytic cycle, the shared substrate pocket should differentially recognize all substrates such as NAD(P)H, CH3-H4folate, and CH2-H4folate, along with their intermediates. The minimum number of unique structures that can accommodate the multiple MTHFR reaction substrates and intermediates and support the multiple catalytic steps are shown in Figure 1C. In the folate-dependent oxidative half reaction, there are at least three distinct folate species that are expected to be recognized and differentially activated in the MTHFR active site  and any other folate-bound intermediate MTHFR structures  have yet to be determined.Crystal structures of MTHFR have been previously determined from various species, including E. coli , T. thermophilus , Haemoph), human , and Sacrevisiae . All MTHtructure . Structuive site  5). In . In E. crmediate C and S1.ifferent . Similar4folate) substrate analog. CHO-H4folate has a formyl group on the N5 position of tetrahydrofolate (H4folate) and was selected for study because it has found use as a nonreactive proxy for CH2-H4folate environment near the folate ligand. Together, our findings suggest that CHO-H4folate induces active site rearrangements as a result of folate-binding, resulting in a unique MTHFR folate-binding mode. We describe the possible functional significance of the CHO-H4folate-binding mode found in tMTHFR and the potential mechanistic role of an observed active site Ser-His-Glu-Thr quartet. MTHFR employs a “Spartan strategy”  activation , an additional shoulder peak was evident around 290 nm after treatment with CHO-H4folate.In this study, we used CHO-Htivation . Indeed,synthase , thymidysynthase , DHFR  use4folate binding indicates that the folate analog can strongly bind to tMTHFR in solution. Subsequently, we wanted to establish the effect of CHO-H4folate binding on tMTHFR activity. Substrate inhibition/competitive substrate inhibition is often observed in enzymes that utilize a ping-pong mechanism. tMTHFR in particular exhibits strong substrate inhibition based on a CH3-H4folate:menadione oxidoreductase assay, which reports on the reverse MTHFR oxidative half reaction  by obstructing tMTHFR•NADH complex formation.The tight CHO-Hreaction . In addireaction , 8, othereaction . Therefo4folate inhibits activity in tMTHFR based on our NADH:menadione oxidoreductase steady-state assay (B). We wanted to determine the mode of inhibition and therefore generated double-reciprocal plots using NADH concentration versus velocity curves. Interestingly, the determined double-reciprocal plots of MTHFR activity at different CHO-H4folate concentrations indicate noncompetitive inhibition (C). This mode of inhibition suggests that CHO-H4folate can bind both to the E(nzyme) and the E(nzyme)S(ubstrate) complex, suggesting that in the reductive half reaction, CHO-H4folate can compete with NADH for binding to the active site pocket but can also bind to the tMTHFR•NADH complex in a mode that we have yet to structurally characterize. To our knowledge, there is no report describing MTHFR inhibition by CHO-H4folate. Based on preliminary experiments, the NADPH:menadione oxidoreductase activity of recombinant human MTHFR  . As the pH is increased, the CHO-H4folate tMTHFR inhibitory effect is less pronounced . The enzyme activity (the NADH substrate consumption per minute) without CHO-H4folate at pH 6.0 was used as a reference point and set as 100%, while the MTHFR activity at pH 7.0 and pH 8.0 was 95% and 120%, respectively. When CHO-H4folate was present at 14 μM , the activity at pH 6.0, 7.0, and 8.0 was 9%, 23%, and 60%, respectively, as compared to the activity in the absence of CHO-H4folate. This suggests that the effect of pH on the enzyme activity is minimal when CHO-H4folate is absent; however, in the presence of CHO-H4folate , the enzyme activity was largely influenced by pH. Our data indicate that solution pH may affect the tMTHFR•CHO-H4folate complex stability and therefore impact activity as determined with our NADH:menadione oxidoreductase assay. In the presence of CHO-H4folate, at low pH, tMTHFR is trapped in a tMTHFR•CHO-H4folate inhibitory complex, which prevents tMTHFR•NADH complex formation because NADH and folate share the same single substrate-binding pocket in MTHFR. As the pH is raised, this tMTHFR•CHO-H4folate inhibitory complex can more readily disassemble, allowing for the formation of a productive tMTHFR•NADH complex.We further investigated the effect of pH on the tMTHFR activity inhibition by CHO-H4folate tightly (A) and that it inhibits tMTHFR activity (B), we contemplated how it may relate to the previously described role of CH3-H4folate as a potent substrate-inhibitor in the CH3-H4folate:menadione oxidoreductase activity assay (4folate complex. The other distinct binding mode is the CH3-H4folate–binding pose found in the eMTHFR•CH3-H4folate structure (productive). We found that tMTHFR•CHO-H4folate complex inhibition is pH-dependent, and therefore we hypothesized that the complex formation of tMTHFR•CH3-H4folate in the CHO-H4folate–binding mode is similarly pH sensitive. If so, the substrate inhibition by CH3-H4folate in the CH3-H4folate:menadione oxidoreductase assay could likewise be pH-dependent.Given that tMTHFR binds CHO-H tightly A and thaactivity B, we conty assay . We prop3-H4folate:menadione oxidoreductase activity was measured at various pHs  is plotted versus CH3-H4folate substrate concentrations, and curves of the CH3-H4folate:menadione oxidoreductase activity in varying pHs are displayed. At pH 6.0, surprisingly, we detected little product formation at concentrations of CH3-H4folate between 0 and 100 μM. In contrast, as the pH is increased, the inhibitory effect is less pronounced , and substrate inhibition was observed only at high CH3-H4folate substrate concentrations (100 μM for pH 8). The pH dependence of the substrate inhibition by CH3-H4folate follows a similar trend to that of NADH:menadione oxidoreductase activity inhibition by CHO-H4folate . It seems that at pH 6.0, almost all tMTHFR•CH3-H4folate forms an inhibitory or nonproductive complex. However, at higher pHs and especially at pH 8.0, the activity is less inhibited, indicating that tMTHFR is active because the enzyme could form a reactive complex with CH3-H4folate. The pH dependence of tMTHFR inhibition (B and D) by CHO-H4folate and CH3-H4folate lends credence to the notion that tMTHFR exists as a conformational ensemble and exhibits conformation sampling , with dC) . We. WeC)  , suggesting that the β-sheet structure of the β8α8 barrel, where the active site is located, is rigid and remains unperturbed  , despiteerturbed . Howeverbind FAD  and S5. C). In the ternary complex of tMTHFR with FAD and CHO-H4folate bound (referred to as “tMTHFR•CHO-H4folate” in this paper), we observe that the folate pterin ring is stacked against the si-face of the FAD isoalloxazine ring. This is similar to the positioning of the CH3-H4folate pterin ring bound to eMTHFR  ternary complexes are presented in stereo mode  and therefore involved in either hydride transfer and/or iminium formation/stabilization  relocating but still engaging, albeit from the side rather than from above, with the PABA ring. Moreover, another aromatic residue, Phe20, in tMTHFR•CHO-H4folate also rearranges, moves closer, and pi-stacks against the PABA moiety from the opposite site of Arg219. As a result, a unique PABA pocket is observed in the tMTHFR•CHO-H4folate structure, in where Phe20 and Arg219 now sandwich and enclose the PABA moiety. The unique protein arrangement around PABA and its functional implications are further discussed in a subsequent section.A close inspection of the active site, as shown in 3-H4folate binding to eMTHFR, Asp120, Lys222, and Phe223 reposition to engage and interact with the CH3-H4folate substrate (D). The local conformational changes in the active site upon binding CHO-H4folate in tMTHFR (E) are even more striking than in eMTHFR (D). In addition to the rearrangement of Asp109, Arg219, and Phe220 , Phe20 and Thr49 are also found in different positions in the tMTHFR•CHO-H4folate structure compared to the eMTHFR•CH3-H4folate one. The repositioning of Thr49 in the tMTHFR•CHO-H4folate structure is of particular note. The Thr49 hydroxyl group hydrogen bonds with the Glu18 side chain in the tMTHFR•CHO-H4folate structure, a residue whose polarity, as previously mentioned, is essential for catalysis . Comparison of the PABA-binding pockets in the tMTHFR•CHO-H4folate and the eMTHFR•CH3-H4folate reveals that these proteins interact with a different set of invariant active site Phe residues  is replaced by one with an Arg residue (Arg219). Furthermore, a different Phe residue  approaches and stacks with PABA ring from the reverse side of the Phe223-contacting side in eMTHFR . The eMTHFR structure in pi interaction between these Phe and Asn residues (Phe30 and Asn278 in eMTHFR). The Phe20 residue is mostly conserved from bacteria to human MTHFRs, with only a few exceptions. The rare example is Leishmania infantum MTHFR, in which the Phe residue is replaced but could be functionally conserved by a Tyr residue . The Asn residue is strictly conserved in MTHFRs. The patient mutation in this conserved Asn residue (Asn324 in human MTHFR) to Ser causes severe MTHFR-deficiency (pi interaction is not present in the holo-monomer of the folate-free dimer mode 2 tMTHFR structure (D).It is intriguing that Phe20 in tMTHFR•CHO-Htructure D.B, are excluded with their space now occupied by Phe20 . Though the loss of a Phe-Asn amide-pi interaction might be slightly deleterious, one could argue that the entropic benefit of a Phe20-lined hydrophobic nook compensates for this loss. The novel PABA pocket also places several aromatic residues near the PABA ring that can pi-stack ; Arg219 (cation-pi) and Met209 (Met-aromatic) provide further stabilization of the PABA moiety. The water rearrangement is of potential significance, since changes in the water environment can influence the flexibility of the loop region (L4) that carries Asp109, which is essential not only for binding but also for activation of folate  anchor the folate molecule. However, the water arrangement near the interface of the folate pterin ring, the FAD isoalloxazine ring, and the L4 loop conformation are different in these two bacterial MTHFR structures (B). In eMTHFR, the L4 loop conformational diversity is dependent on ligand-binding and pH , and L4 is in the closed conformation . The pterin and PABA in CH3-H4folate are almost orthogonal, as indicated by the approximately right, 90∼93° angle they form, whereas those in CHO-H4folate are arranged in a more acute fashion/manner with an 84∼86° angle (C-1 and C-2). As a result, the distances between N10 and C11 are 4.1 Å and 3.5 Å in CH3-H4folate and CHO-H4folate, respectively (C-1 and C-2). Guided by the shorter N10-C11 distance in CHO-H4folate, a hypothetical conformation of the CH2-H4folate substrate was generated using the CHO-H4folate–binding mode as a mold to roughly approximate the position of the carbon attached to the N5 of folate in Figure 4C-3. In this model, the N10-C11 distance in CH2-H4folate is 1.5 Å, indicating that the novel binding mode of CHO-H4folate might represent a folate-binding mode more akin to that of CH2-H4folate or an intermediary state prior to its formation, given that its N11-C10 distance is in between that of CH3-H4folate and CH2-H4folate.The open L4 loop as well as the new/unique PABA pocket in tMTHFR•CHO-Hdihedral C. The ptC), CH2-H4folate, (C-5), after or concurrent to a protonation event, is converted to an iminium intermediate (C-6), followed by hydride transfer from FAD to the iminium intermediate to form CH3-H4folate (C-7). In the hydride transfer reaction (C-6 and C-7), a hydride needs to be shuttled between the N5 FAD atom and the N5-C atom of folate. Therefore, the two ligands (FAD and folate) in the active site of MTHFR are in close proximity and oriented appropriately so as to enable electron transfer. The catalytically essential Glu residue , N5-C of folate, and N5 of FAD need to be in close proximity for hydride transfer in the folate-dependent reaction of MTHFR to occur. Indeed, the distance between N5-C of folate and N5 of FAD in the eMTHFR•CH3-H4folate structure is 3.3 Å (4folate structure is 3.8 Å in one monomer and 4.0 Å in the other (4folate structure indicates that the ligands and active site residues are not in an arrangement (as) conducive to hydride transfer. The catalytically essential Glu residue (3-H4folate) is situated in between and in close proximity to both pterin and isoalloxazine rings . For example, the FAD N5 atom in tMTHFR is 3.8 Å apart from Glu18 Oε2, while in eMTHFR, it is 4.8 Å away from Gln28-Nε2 (the Glu residue is mutated to Gln)  . Althoug4folate structure, the “open” state of the L4 loop reconfigures the hydrogen-bonding network between Asp109, folate, and FAD (A) slightly separating the folate pterin ring and FAD and disrupting an Asp109 bridging hydrogen network. Incidentally, another unique hydrogen-bonding network in the tMTFHR•CHO-H4folate structure arises from the rotation of Thr49 and its interaction with Glu18 to create a Ser-His-Glu-Thr quartet as shown in 4folate structure form a novel Ser-His-Glu-Thr quartet. Interestingly, the Ser-His-Glu triad is highly conserved in MTHFR, and its hydrogen-bond network further extends from the hydroxyl group of Ser16 to a surface water through an internal water molecule and the backbone peptide bond between Leu14 and Phe15  and the holodimer tMTHFR structure at low resolution   are lengthy, at more than 3.5 Å. Thus, the Thr-Glu interaction in those structures is either weaker than that in the tMTHFR•CHO-H4folate complex or does not exist. Although the 1B5T and 3APY structures have been determined at near-natural pH, the quartet-containing tMTHFR•CHO-H4folate structure was solved at pH 4.5. However, a tMTHFR structure solved at pH 7.8  CH2-H4folate ring opening  provides a possible clue as to how CHO-H4folate or CH3-H4folate can act as inhibitors of tMTHFR activity. We postulate that there is an ensemble of two folate-accessed conformations and that their relative population is affected by pH. Protein protonation can be responsible for the suggested conformational changes, although the nature of the amino acid residue(s) that is(are) protonated could not be identified in the present study.The pH dependence of tMTHFR inhibition , B and C3-H4folate substrate inhibition that we observe, there are no available data to support that MTHFR activity is regulated by a second folate-binding site. We have found no crystallographic or conclusive biochemical data to suggest that folate binds anywhere else but in the active site. We therefore hypothesize that the observed folate-binding mode in our tMTHFR•CHO-H4folate structure is related to the observed inhibition by CHO-H4folate and by extension the substrate inhibition by CH3-H4folate. It is possible that at low pH, a large fraction of the enzyme is trapped as the inhibitory/nonproductive complex with CH3-H4folate, resulting in little to no turnover; higher pH would allow the inhibitory complex to disassemble or convert to the deprotonated tMTHFR form, relieving the observed substrate inhibition. Our model in where CH3-H4folate can access two pH-dependent folate-binding poses (or two protein ensembles) is consistent with the observed substrate inhibition based on the CH3-H4folate:menadione tMTHFR oxidoreductase assay. As previously mentioned, tMTHFR activity is much more sensitive to CHO-H4folate inhibition than human MTHFR. A possible explanation for the extreme sensitivity of tMTHFR to CHO-H4folate is its high isoelectric point (pI). While the calculated tMTHFR pI is 9.0  at pH 6.7 is 19 μM, which is substantially lower than that at pH 7.2, 88 μM  per se are not potent factors in isolation, but they become strong effectors when they cooperate/potentiate their respective effects. Despite binding in the active site, CHO-H4folate was found to be a noncompetitive inhibitor, causing a ligand-induced conformational change from dimer mode 2 to dimer mode 1. A similar effect was found previously, caused by FAD loading, where the change from a half-FAD occupancy (dimer mode 1) to an FAD-replete dimer would promote an identical shift in dimer interfaces . Such PABA-ring–driven folate conformational change was previously proposed . Therefore, we posit that the active site conformation in the tMTHFR•CHO-H4folate structure represents a different folate functional state, either the substrate CH2-H4folate or the ring-opening folate intermediate-binding mode (C-5 and C-6 respectively). In the mode represented by the CHO-H4folate–binding pose, an active site remodeling is manifested by the combination of the Phe20-containing PABA pocket arrangement, an open conformation of the L4 loop that affects pterin folate positioning, and the Ser-His-Glu-Thr quartet formation that allows for modulating the Glu reactivity. In sum, the unique folate-binding mode and structural active site remodeling in tMTHFR•CHO-H4folate structure compared to the eMTHFR•CH3-H4folate one are potentially indicative of how MTHFR binds and activates CH2-H4folate or the subsequent ring-opening iminium intermediate. Our hypothesis would be able to successfully rationalize the MTHFR biochemical properties of previous reports (C-7).In the tMTHFR•CHO-H and FAD  and 5, wpport it C-7. Ther reports , 30 as w reports C-7.4folate and its noncompetitive inhibition in the NADH:menadione oxidoreductase assay were unexpected, as the change in dimer interface (dimer mode 1) is less extensive (than dimer mode 2); crystallographic evidence shows that CHO-H4folate is found only in the active site. Several other folate-binding enzymes, namely DHFR . The subtle but purposeful active site changes in MTHFR represent a rather impressive biological example and an adept demonstration of how intricate dynamic changes grant a single enzyme the ability to bind, stabilize, transform, and activate different substrates, using a delicate rearrangement of its active site to perform radically different chemistries. Despite increasing recognition of its important role in human health, the same features that enable MTHFR to bridge two metabolic cycles, namely its structural flexibility, have also hindered attempts to study its activity at the molecular level. Even so, the use of bacterial homologs and folate analogs have allowed one to trap MTHFR in action; simultaneously elegant and deceptively simple, our MTHFR work demonstrates Nature’s creativity as a “chemist”, providing us with lessons on how regulatory control can arise from local dynamic structural changes in the active site.MTHFR provides a paradigm of how structurally dynamic changes/conformational sampling, induced by both ligand-binding and/or pH, can allow a single active site to accommodate chemically distinct species (in NADH and at least three different folates). MTHFR is able to perform chemically distinct transformations, including but not limited to carefully controlled redox chemistry and a difficult C-N bond/ring cleavage, acting as the sole source of a folate-based methyl donor. Using the same set of limited amino acids, MTHFR can perform the reaction 6RS)-5-CHO-H4folate , NADH , and menadione (Sigma M5625) were purchased from Sigma. (6S)-5-CH3-H4folate  and FAD  were purchased from Eprova (Merck Eprova AG) and Usb corporation, respectively.(wt-H) (Glu18Gln-H) were used to produce recombinant protein with an uncleavable hexa-Histidine tag on the C-terminus. E. coli BL21star(DE3) was transformed with the expression vector. Protein was induced in autoinduction media   and pET(on media . Cells wE. coli cells were suspended in 50 mM potassium phosphate buffer pH 7.4 (KPB) containing 0.2 M sodium chloride (NaCl) and then sonicated. The cell lysate was centrifuged, and the supernatant was pooled. After addition of free FAD, the supernatant was incubated at 70 °C for 15 min. Clear supernatant was obtained by centrifugation and filtration. The heat-treated, clear extract was loaded onto an Ni-affinity column , which was equilibrated with 50 mM KPB, 0.2 M NaCl, and 50 mM imidazole. After washing the column with 50 mM KPB, 0.2 M NaCl, and 100 mM imidazole, the target protein was eluted with 50 mM KPB, 0.2 M NaCl, and 300 mM imidazole. The eluate was dialyzed against 0.1 M KPB, 0.1 M NaCl at 4 °C overnight. The dialyzate was concentrated and stored at 4 °C.Protein purification was performed as previously reported . E. coliEnzyme assays were performed as previously described, using three distinct batches of purified enzyme to act as biological replicates . NADH:me3-H4folate:menadione oxidoreductase activity was determined as follows (6S)-CH3-H4folate was added. The total volume of the reaction was 800 μl. The reaction was quenched by adding 200 μl of a 13 M formic acid and 5 M hydrochloric acid mixture, followed by heating at 90 °C for 10 min to convert the CH2-H4folate product to methenyltetrahydrofolate. After cooling, absorbance at 350 nm was measured. To calculate the concentration of methenyltetrahydrofolate, the molar absorption coefficient of 26.0 × 103 was used. The following equation ), equilibrated with 50 mM KPB, pH 7.4 to remove free ligand. CHO-H4folate coeluted with tMTHFR from the gel filtration column, which was equilibrated with free CHO-H4folate buffer, indicating that tMTHFR with oxidized FAD forms a tight complex with CHO-H4folate. Comparison of the UV-VIS spectra, of the free CHO-H4folate versus protein-bound CHO-H4folate after gel filtration, at 290 nm , allowed us to estimate that tMTHFR was approximately 75% replete. When crystals of the tMTHFR•CHO-H4folate complex were prepared, they grew under the presence of added excess CHO-H4folate in the crystallization solution. Electron density corresponding to CHO-H4folate was found only in the active site of tMTHFR, suggesting that the stoichiometry ratio of CHO-H4folate to a tMTHFR monomer is 1:1.Purified tMTHFR was treated with an excess amount of CHO-Hwt crystals were grown as follows. FAD-replete tMTHFR (25 mg/ml) in 0.1 M KPB and 0.1 M NaCl was mixed in a 1:1 ratio with a reservoir solution . Crystals were grown at 20 °C using the sitting-drop vapor diffusion method.Folate-free tMTHFRGlu18Gln crystals were grown as follows. As-purified tMTHFRGlu18Gln was prepared in a final concentration of 13 mg/ml in 0.1 M KPB, 0.1 M NaCl, and 0.6 mM of CH3-H4folate and was then mixed in a 1:1 ratio with reservoir solution . The crystals were grown at 20 °C using the hanging-drop vapor diffusion method. Crystals were transferred to a harvesting solution (the reservoir solution containing ≈20% glycerol) for 1 min and then flash-frozen in liquid nitrogen.Folate-free tMTHFR4folate complex was obtained by cocrystallization. Specifically, FAD-replete tMTHFR at a concentration of 28 mg/ml and ∼77% CHO-H4folate replete was used. The protein solution was diluted to 20 mg/ml with 0.1 M KPB, 0.1 M NaCl, and 1.2 mM CHO-H4folate. Protein solution was then mixed in 1:1 ratio with a reservoir solution  at 4 °C. Crystals were transferred to a harvesting solution (the reservoir solution containing ≈20% glycerol) for 1 min and then flash-frozen in liquid nitrogen. Diffraction data for folate-free MTHFRwt and the tMTHFR•CHO-H4folate complex were collected on the GM/CA-CAT 23-ID-D beamline, while diffraction data for folate-free MTHFRGlu18Gln was collected on the LS-CAT 21-D-D beamline at the Advanced Photon Source, Argonne National Laboratory. Crystals were mounted under gaseous nitrogen at 100 K. X-ray data sets were recorded by a Pilatus 3-6m detector for the former and on an Eiger 9M detector for the latter, respectively.The tMTHFR•CHO-HGlu18Gln data set, which was processed by DIALS  (S)-CHO-H4folate stereoisomer could be appropriately assigned to the electron density in each monomer, despite using the (6RS)-CHO-H4folate racemic mixture when preparing the complex with tMTHFR. Though tMTHFRGlu18Gln was cocrystallized with CH3-H4folate and the presence of a small-molecule was found in the active site near FAD, partial occupancy/nondefinitive electron density did not allow for a clear ligand assignment. For manual model building, COOT   in the Cng, COOT  was usedng, COOT . All figng, COOT .Table 1X7TH4 (DOI: https://doi.org/10.2210/pdb7TH4/pdb), 7TH5 (DOI: https://doi.org/10.2210/pdb7TH5/pdb), 8EAC (DOI: https://doi.org/10.2210/pdb8eac/pdb)Structural data are deposited in the RCSB Protein Data Bank. PDB-ID-codes: This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Gowan et al. Nature Communications 10.1038/s41467-021-21469-w (2021)To reconstruct past sea levels, for estimating changes in global ice volume, it is necessary to obtain evidence from biofacies that rely on in situ fossil material and that grew at specific, shallow-water depths close to the oceans shorelines during their life span. At such sites, a time series is also necessary for estimating the exact timing of the lowest sea level by determining geological sequences and the dating has to be made on the in situ fossil organisms themselves.1 (hereafter EJG21) presented a new global ice sheet reconstruction for the last 80,000 years spanning the time period of the Last Glacial Maximum (LGM: around ca. 20 ka) and marine isotope stage 3 (MIS3: 57–30 ka). The reconstructed global ice volume relied on a simple ice model constrained by near-field ice volumes derived from glacio-geological evidence. The two main conclusions arising involved the magnitude of the global mean sea level during the LGM and during MIS 3. Firstly, their LGM sea level at −116 m is significantly shallower than previously reported values 5. Secondly, EJG21’s MIS3 sea level is also much shallower than most other records suggest  and geochemical analyses4 of the 583 cm core KH11-1GC6 confirmed a local LGM sea level of −120 m  is −118 m  by 20–80 m than previously reported. Important constraints for the MIS 3 GMSL come from both uplifted and submerged coral reef terraces in Papua New Guinea (PNG), a tectonically-active site, that has been extensively studied with corals from terrace surfaces and drill cores and were mass-spectrometric uranium/thorium dated  Expedition 310 also indicate GMSL at −65 to −75 m12. These results contradict those tabled by EJG21 . Unfortunately, the EJG21 study leads to an additional and unnecessary confusion rather than to a convergence of views.In conclusion, the paper by EJG21 argued that they have solved the missing ice problem of the LGM


Mage = 21.63 years old, SD = 2.12) played one of three games: puzzle (n = 19), TBS (n = 24) or RTS (n = 24) for 4 weeks on their smartphones. Participants completed three inhibition tasks, working memory (WM), and had their tympanic membrane temperature (TMT) taken from each ear before and after playing the games. Results showed that only the puzzle game group showed an improved response inhibition while controlling for WM. There were no significant changes in the distractor inhibition tasks. We also found that there was an increase in left TMT while playing RTS, suggesting the presence of increased impulsivity in RTS. Our findings suggest that puzzle games involving logical contradiction could improve response inhibition, showing potential as a tool for inhibition training.Past studies have shown evidence of transfer of learning in action video games, less so in other types, e.g. strategy games. Further, the transfer of learning from games to inhibitory control has yet to be examined from the perspectives of time constraint and logic contradiction. We examined the effect of strategy games  on inhibition (response inhibition and distractor inhibition) and cerebral hemispheric activation over 4 weeks. We predicted that compared to RTS, puzzle and TBS games would (1) improve response and distractor inhibition, and (2) increase cerebral hemispheric activation demonstrating increased inhibitory control. A total of 67 non-habitual video game players ( We propose two possibilities to explain the transfer of inhibition; time constraint and logic contradiction.The transfer of learning and skill improvement from a training task to an untrained task could occur when the tasks use similar skills or processing patterns Taatgen, . PlayingWhen playing a game, the available time becomes a variable in determining how players play the game. RTS games are fast-paced, requiring players to respond quickly and adaptively in real time. In contrast, puzzle games and TBS games generally do not have time constraints, allowing players to take time in planning their moves by simulating, evaluating, and revising the possible options to identify optimal solutions among many possible suboptimal or incorrect moves. This suggests that when given time, inhibition would be involved in inhibiting the selection of incorrect moves during planning  have a limited number of optimal solutions and (2) are designed with logical contradictions that lure players into making incorrect assumptions Brown . AlthougUnlike puzzle games, RTS and TBS games do not have logical contradictions in the game design. This could be attributed to the multiple optimal solutions in RTS and TBS games. The stages in these games do not have a single optimal solution, which encourages players to adapt and make decisions from a large number of possibilities. For example, in the TBS game Warlords of Aternum  is a possible factor in inhibition performance. Past studies have shown that having better WM is associated with better performance in RTS games , where the difference between right TMT and left TMT (ΔTMT) reflects the difference in hemispheric activation. Individuals with greater inhibition were found to have a more positive ΔTMT (right TMT minus left TMT), indicating a greater right hemispheric activation than the left hemispheric activation Helton, . In contOur overarching aim was to examine the effects of types of strategy games on response and distractor inhibition, as well as hemispheric activation. To our knowledge, the comparison of time constraint and logic contradiction found in games has yet to be examined. Further, having higher WM is associated with improved inhibition, suggesting that WM may be a confound on inhibition performance. Based on the literature above, we hypothesised that (H1) there would be a greater improvement in response and distractor inhibition in the puzzle group and TBS group compared to the RTS group, and (H2) there would be an increase in ΔTMT in the puzzle group and TBS group compared to the RTS group.n = 36), TBS (n = 36), and puzzle (n = 33) conditions. Inclusion criteria included (1) were non-habitual video game player (NVGP) with playing less than 1 h of video games per week in the past 12 months or casual player with playing less than 1 h of RTS, TBS, and puzzle games and less than 3 h of any other games (e.g. simulation games) per week in the past 12 months, (2) aged between 19 and 40 years old, (3) owned a smartphone, (4) right-handed or ambidextrous, (5) had normal or corrected to normal vision, and (6) did not have a confirmed diagnosis of psychiatric condition, neurological condition, or substance abuse. After removing some attrition (n = 35), our final sample was 67 participants . Our participants were mainly female (89.5%) with a mean age of 21.63 years (SD = 2.12)  and they were randomly assigned to 1 game category (e.g. RTS) to which they played and reviewed 4 RTS games for an hour per game in a week. The four games were selected based on the following criteria: (a) the game description fits the game type ; (b) the game had at least 4.3 stars ratings with 40,000 reviews on Google Play Store as of 21st February 2019; (c) the game consisted of single-player option and progressed in stages; and (d) the game was available for free on android phones and iOS phones. After playing the games, we selected those with the highest mean ratings on perceived strategic thinking and game experience for each game from our testers  = 1.51, p = 0.249, and strategy, F = 2.70, p = 0.093.For game selection, we recruited 21 young adults , and the Stroop task and the Multi-Source Interference Task (MSIT), both to measure distractor inhibition. All tasks were completed on a Windows computer (with a 60 Hz frame rate) in the research laboratory.Stop-Signal Task. We used the stop-signal task on the STOP-IT software  for each participant at 50% to produce the most reliable inhibition estimates. A fixation cross was displayed for 250 ms between each trial. Stop-signal task performance was measured using the stop-signal reaction time (SSRT). SSRT was calculated from correct responses using the following formula: SSRT = mean of go-RT  minus the mean of SSD. A lower SSRT indicates greater stop-signal task performance, which reflects greater response inhibition.The colour Stroop task was programmed on Psychopy version 1.90.1  was used to measure the TMT. Three TMT readings were recorded on each ear immediately before and after each of the inhibition tasks . ΔTMT was calculated as the average difference in TMT between right ear and left ear (ΔTMT = Σ(right TMT—left ear TMT)/18). A more positive value of ΔTMT indicates higher right TMT relative to left TMT, which reflects greater cerebral activation in the right hemisphere compared to the left hemisphere.Working Memory. WM was measured using the operation span (OSPAN) task on the CogLab software  to seven (very true). Higher average scores on these measures indicate greater game satisfaction, perceived similarity, and perceived strategic thinking.Participants completed questions on game satisfaction, perceived similarity, and perceived strategic thinking. We measured game satisfaction using six subscales of the Game User Experience Satisfaction Scale (GUESS) on a 7-point Likert scale with one ‘strongly disagree’ to seven ‘strongly agree’ . Next, we conducted trial-level data cleaning to ensure all responses were above the threshold of anticipatory responses (i.e. 250 ms) and below the maximum response duration on each task . We then analysed the data for unreliable data and outliers at the participant level using 5% trimmed means and boxplots. We also used Shapiro–Wilk tests and scatterplots for normality, homogeneity of regression slopes, and homoscedasticity; Levene’s tests were conducted to check for the homogeneity of error variances. We also examined whether WM was a covariate in the effect of type of game on inhibition by conducting bivariate correlations between WM and all measures of inhibition. We then used analysis of covariance (ANCOVA) to test our hypotheses. Simple main effect analysis was conducted to follow-up on significant interaction effect. Speed-accuracy trade-off was also examined for significant interaction effect.We first screened participants’ game progress logs to ensure that participants have completed 20 h of training with at least 10 sessions as well as each training session was at least 30 min and did not exceed 2 h to ensure that participants followed our training guidelines. Participants’ total time spent playing the assigned game and time spent playing in each sitting were not significantly different across groups (ps < 0.01). Therefore, we included WM as a covariate in examining response inhibition and distractor inhibition.From our correlation analysis, our results showed that WM was negatively correlated with stop-signal task performance and Stroop task performance, but not MSIT task performance or ΔTMT  = 5.34, p = 0.025, partial η2 = 0.09 indicating that WM exerted its influence on SSRT performance. The main effect of type of game was not significant, but SSRT significantly decreased from pretest  to posttest . The interaction effect was also significant, F = 3.93, p = 0.026, partial η2 = 0.13  × 2  mixed ANCOVA was conducted with WM as a covariate. The effect of WM on SSRT was significant, F, 54 = 5.F = 5.73, p = 0.027, partial η2 = 0.23, but not in the RTS group and the TBS group (ps > 0.05).We followed up with one-way repeated measures ANCOVAs. SSRT significantly improved in the puzzle group, F = 4.39, p = 0.041, partial η2 = 0.08. The interaction effects and the main effects were not significant . Overall, the results indicated no evidence for speed-accuracy trade-off on the stop-signal task performance  while controlling for WM. A speed-accuracy trade-off would be indicated by a lower go-trials RT and a lower accuracy only in the puzzle group. We only found a significant effect of WM on go-RT, F1, 5 = 4.39, F = 4.59, p = 0.014, partial η2 = 0.14, similar to when WM was a covariate. However, the simple main effect analysis was different without WM as a covariate. Here, we found that participants in the RTS group showed a significant improvement from pretest  to posttest , F = 9.25, p = 0.008, partial η2 = 0.38. In contrast, when WM was included as a covariate, the RTS group showed a non-significant improvement  × 2 (Time) mixed ANOVA on SSRT where WM was not included as a covariate. The interaction was significant when WM was not a covariate, F1, 6 = 4.59,F = 4.79, p = 0.033, partial η2 = 0.07. We did not find a significant interaction effect or main effects of time and game type   = 2.03, p = 0.160. We did not find significant interaction effect or main effects on game type and time   = 4.19, p = 0.020, partial η2 = 0.13. However, we did not find significant main effects of time, F = 0.74, p = 0.394, partial η2 = 0.01, and game type, F = 0.22, p = 0.805, partial η2 = 0.01  × 2 (Time) mixed ANOVA. Results showed that the interaction effect on ΔTMT was significant, F = 11.25, p = 0.004, partial η2 = 0.43, but not in the puzzle group, F = 0.06, p = 0.815, partial η2 = 0.00, and the TBS group, F = 2.67, p = 0.118, partial η2 = 0.12.Simple main effect analysis was conducted using one-way repeated measures ANOVAs as follow-up to the interaction effect. ΔTMT significantly decreased in the RTS group, F = 0.80, p = 0.454. There was significant difference for game satisfaction, F = 7.93, p = 0.001, partial η2 = 0.20, to which RTS game was rated significantly lower in-game satisfaction compared to the TBS game (p = 0.003) and the puzzle game (p = 0.002). As for perceived similarity, there was no significant difference for stop-signal and MSIT, both ps > 0.864. However, the perceived similarity for the Stroop task was significantly different across groups, F = 3.69, p = 0.031, partial η2 = 0.11, to which puzzle game was rated significantly more similar to the Stroop task than the RTS game (p = 0.031).We also analysed game satisfaction, perceived similarity, and perceived strategic thinking using one-way between-subjects ANOVA (see Table We hypothesised that the absence of time constraints (i.e. more planning time at leisure) in strategy games would be beneficial in training response inhibition, and our results showed that this was the case only for the puzzle game after controlling for WM. Our findings are concordant with Oei and Patterson’s  findingsWe did not find evidence of response inhibition improvement in the TBS game, another game with no time constraint similar to the puzzle game. One possibility is the presence of logical contradiction, commonly found in puzzle games but not in TBS games. Games with logical contradiction would train inhibition further, as ‘mistakes’ would lead to incorrect moves and encourage players to inhibit their impulsiveness. This is because puzzle game often leads players into making incorrect assumptions, thus they have learned to inhibit their assumptions to make counterintuitive moves.Unlike response inhibition, we did not find any significant difference in the distractor inhibition tasks. These strategy games may require players to utilise all information and options provided on the user interface and the playing field for optimal moves or completion of the goal, thus requiring them to remain attentive to all visual cues instead of inhibiting non-relevant stimuli. To perform optimally in the RTS game accurately, players would need to accurately keep track of enemy units and abilities while managing their own units and abilities in real-time. This improvement in attention to peripheral cues could be transferred to visual attention tasks such as multiple object tracking but not distractor inhibition  increased relative to the right TMT (indicative of right hemispheric activation), suggesting a decrease in inhibition. Why is this so? One reason could be an increase in impulsivity and risk-taking. Individuals with greater impulsivity and risk-taking tendencies have been shown to have more negative values of ΔTMT (i.e. higher left hemispheric activation)  rated the RTS game lower in-game satisfaction compared to the TBS and puzzle games. Participants also commented that they did not enjoy playing or had difficulty understanding to play the assigned game. Non-habitual players may have had a more difficult time in understanding the technical gameplay of the Galaxy Reavers RTS game (Good Games LLC, Our findings suggest that puzzle games could be used to train response inhibition. Clinicians could explore the use of puzzle games in training individuals with impaired response inhibition, such as older adults (Basak et al., In conclusion, when controlling for WM, we found that only puzzle games improve response inhibition, suggesting that planning in puzzle games could train players in inhibiting their preplanned responses. The effect was not significant for distractor inhibition, suggesting that changes in one measure of inhibition are not necessarily accompanied by a change in another. RTS game increased left hemispheric activation relative to right hemispheric activation, which is indicative of increased impulsivity and risk taking. Future research could further examine the planning process in these games and their potential in inhibition training.


We present experimental and numerical studies demonstrating the influence of geometrical parameters on the fundamental spin-wave mode in planar 1D magnonic crystals. The investigated magnonic crystals consist of flat stripes separated by air gaps. The adjustment of geometrical parameters allows tailoring of the spin-wave frequencies. The width of stripes and the width of gaps between them affect spin-wave frequencies in two ways. First, directly by geometrical constraints confining the spin waves inside the stripes. Second, indirectly by spin-wave pinning, freeing the spin waves to a different extent on the edges of stripes. Experimentally, the fundamental spin-wave mode frequencies are measured using an all-optical pump-probe time-resolved magneto-optical Kerr-effect setup. Our studies address the problem of spin-wave confinement and spin-wave dipolar pinning in an array of coupled stripes. We show that the frequency of fundamental mode can be tuned to a large extent by adjusting the width of the stripes and the width of gaps between them. They can act as waveguides carrying the information encoded in spin waves6. Recently, many publications have focused on the spin waves in the sets of stripes, arranged in-plane periodically  or quasiperiodically . The stripe systems act not only as waveguides. By adjusting their dimensions, we can tailor the spin wave propagation and thus process magnonic signals17.Ferromagnetic stripes are one of the basic building blocks of magnonic structures18. However, this effect is partially mitigated by the reduction of precession amplitude close to the lateral faces for flat stripes, which is called dynamical pining of magnetization20. In other words, the system partially trades the demagnetizing energy related to the presence of surface charges on lateral faces (and related to the presence of dynamic stray field) for other contributions of demagnetizing energy, resulting from the volume magnetic charges induced by the non-homogeneous distribution of the spin-wave amplitude21. The pinning partially confines the fundamental mode within the stripes and causes a frequency up-shift when the stripe’s widths are narrowed. Another effect is the weakening of dipolar coupling between the stripes due to dynamic magnetization pinning because of the reduction of dynamic stray field.The collective magnetization dynamics in stripe arrays depend on the magnetic coupling between the stripes. Thus, the simplest possible structure of this type is a periodic sequence of flat stripes separated by air gaps, which can be carved in thin ferromagnetic films. In such a system, the coupling is provided by a dynamic stray field within the gaps. The strongest dynamic stray field is created by the fundamental mode, where the magnetization precesses in phase. The largest surface magnetic charges and, thus, the strongest dynamic stray field appears on the lateral faces between the stripes, which is also reflected in the ellipticity of precession20: The strength of the pinning is expressed by the pinning parameter22. Then, the pinning can be introduced by a different mechanism: the presence of surface magneto-crystalline anisotropy22. In the general case, the dipolar pinning can be combined with the influence of surface anisotropy20. However, for thin stripes , the dipolar interactions significantly impact pinning20.In the absence of the dipolar interaction, the spin wave is free on the surfaces  models with dipolar and exchange interactions taken into account31.When the dipolar interactions prevail over the exchange interactions, spin wave modes in confined geometry  can be calculated by solving the integral eigenvalue problem with the magnetostatic Green’s function as an integral kernel33, the time-resolved magneto-optical Kerr effect (TR-MOKE) spectroscopy37, to determine the fundamental mode frequency. Using the TR-MOKE technique, we measure the frequency of the fundamental spin-wave mode  on spin wave pinning and consequently on the frequency of the fundamental mode in such structures. Spin wave dynamics is calculated numerically by solving the linearized Landau-Lifshitz equations. We use an experimental technique complementary to Brillouin Light Spectrometry38), and theoretical approach  we used. We also provide the Supplementary Information, which describes: the details of post-processing for the TR-MOKE signal, the semi-analytical calculations of the demagnetizing factors for fundamental mode, and the determination of equilibrium orientation of static magnetization.The paper is organized in the following way. In “Results and discussion”, we present and describe the results of our measurements and computations. We show the dependence of frequency and mode profile of fundamental mode  on the structure’s geometry—stripes width and size of the air gap. “Results and discussion” is finalized with the conclusions. The following section (“Methods”) describes the experimental methods , numerical techniques (finite element method (FEM)The systems consist of CoFeB stripes of finite width mentclass2pt{minimIn our studies, we fixed the thickness mentclass2pt{minimThe numerically calculated FMR frequency for the CoFeB layer is equal to 13.6 GHz. The FMR frequencies obtained experimentally by TR-MOKE are in the range of 13.6 up to 13.95 GHz. Differences may be due to the quality of the samples measured, the quality of the laser in the pump-probe experiment, and other factors. It follows that measurement uncertainties of half a gigahertz are expected, even for simple structures. The value calculated for uniform CoFeB film is marked in Fig. After these preliminary studies, we performed (by TR-MOKE) the measurements of magnetization dynamics in all the fabricated periodic structures with stripes of the widths entclass1pt{minimaIt can be seen that we have achieved good agreement between experimental, computational, and semi-analytical results. Experimental results are subject to significant measurement error even for uniform CoFeB films due to the quality of the CoFeB material (homogeneity of composition), measurement uncertainty , which we have already described for the layers. In the case of periodic structures, the experimental measurement results are subject to even more significant uncertainty due to the difficulty of producing ideal thin magnetic stripes with sharp edges, separated by air gaps of the same width along the length of the stripe. Taking this into account, the presented experimental results are within the measurement uncertainty limit. It can be seen that the fundamental mode frequencies depend on the air gap separations between stripes. The change of fundamental mode frequency between uniform film and periodic structures is continuous. For very small separations, the fundamental mode frequency of periodic structure tends to be the FMR frequency of a uniform film, being the bottom limit. The bigger the separations, the higher the frequency of the fundamental mode of the structure. The fundamental mode frequency is the highest for completely separated stripes—i.e., single stripes—which is the upper limit. In our measurements, it is impossible to measure a signal from a single stripe since the signal would be too small, so the upper limit was only calculated numerically (dash-dotted line) and semi-analytically (dotted line). The fundamental mode frequency of a single stripe is presented as a dotted (dash-dotted) line for each subfigure in Fig. We can notice in Fig.  that theg, i.e., from For a better understanding and more explicit interpretation of the experimental results, we performed the simulations for the extended range of the widths of air gaps We have plotted the profiles of the fundamental mode for systems composed of stripes of one selected width (The profile marked with the label (s) is the profile of the mode of a single 500 nm wide stripe . It is worth noting that the wider stripes are more strongly coupled compared to the narrower stripes, at the same gaps, because the weaker pinning means the stronger stray field produced at the lateral faces of the steps, which is responsible for coupling. (ii) The dipolar interactions between the constituting nanoelements of magnonic planar nanostructure must be considered even if the distance between successive stripes is within a single micrometer. (iii) The TR-MOKE measurements allow for the investigation of the frequency of fundamental mode, i.e., the mode precessing uniformly in space (with wave vector We can summarize our research on the magnetization dynamics in the array of flat stripes arrays by highlighting the following conclusions. (i) The fundamental mode frequency increases with the stripes’ separation. It also depends on the width of the stripe due to the pinning. The frequency of the fundamental mode is lower for wider stripes. This decrease results not only from the extension of the space on which the mode is spanned  but also from the reduction of pinning  at a repetition rate of 40.Magnetization dynamics for precession frequency determination is measured in a time-resolved magneto-optical Kerr-effect setup (TR-MOKE) in an all-optical pump-probe scheme, depicted in Fig. mentclass2pt{minimThe dynamics is triggered via a broadband excitation by depositing energy from the pump beam laser pulses at fluences adjusted to 18:41. Both the magnetization of the ferromagnet 42:The magnetization dynamics in a magnetic system is described by the equation of motion of the magnetization, called the Landau–Lifshitz equationDipolar field y-coordinate. We consider monochromatic SWs in a fundamental mode (f is the frequency).To observe the TR-MOKE signal, we applied the external field mentclass2pt{minimLastly, coupling the Landau–Lifshitz equation with Gauss law for magnetism, we arrive at the following system of equations for the dynamic magnetization 38 to solve8 Eq.  for the flat stripes (Semi-analytical calculations supplemented the numerical results. The fundamental mode frequency was calculated using the Kittel formula for the planar system with the uniaxial shape anisotropy: Supplementary Information.


Necroptosis is a caspase-independent, pro-inflammatory mode of programmed cell death which relies on the activation of the terminal effector, MLKL, by the upstream protein kinase RIPK3. To mediate necroptosis, RIPK3 must stably interact with, and phosphorylate the pseudokinase domain of MLKL, although the precise molecular cues that provoke RIPK3 necroptotic signaling are incompletely understood. The recent finding that RIPK3 S227 phosphorylation and the occurrence of a stable RIPK3:MLKL complex in human cells prior to exposure to a necroptosis stimulus raises the possibility that additional, as-yet-unidentified phosphorylation events activate RIPK3 upon initiation of necroptosis signaling. Here, we sought to identify phosphorylation sites of RIPK3 and dissect their regulatory functions. Phosphoproteomics identified 21 phosphorylation sites in HT29 cells overexpressing human RIPK3. By comparing cells expressing wild-type and kinase-inactive D142N RIPK3, autophosphorylation sites and substrates of other cellular kinases were distinguished. Of these 21 phosphosites, mutational analyses identified only pT224 and pS227 as crucial, synergistic sites for stable interaction with MLKL to promote necroptosis, while the recently reported activation loop phosphorylation at S164/T165 negatively regulate the kinase activity of RIPK3. Despite being able to phosphorylate MLKL to a similar or higher extent than wild-type RIPK3, mutation of T224, S227, or the RHIM in RIPK3 attenuated necroptosis. This finding highlights the stable recruitment of human MLKL by RIPK3 to the necrosome as an essential checkpoint in necroptosis signaling, which is independent from and precedes the phosphorylation of MLKL. Necroptosis is a lytic, caspase-independent, pro-inflammatory form of programmed cell death –4. The tNecroptosis can be induced by the activation of TNF receptors , or variThe precise mechanism of necroptosis signaling remains incompletely understood. As we recently reviewed, the necroptosis pathway is heavily regulated by post-translational modifications . The autHere, we sought to identify (auto)phosphorylation sites of human RIPK3 using phosphoproteomics. By overexpressing human RIPK3, an abundance of phosphopeptides was obtained, from which 21 unique RIPK3 phosphorylation sites were identified. By comparing cells expressing wild-type RIPK3 with that carrying the kinase-inactive D142N mutation, we identified autophosphorylation sites as well as the substrates of other cellular kinases. Extensive mutagenesis analyses revealed that phosphorylation of only two of the 21 RIPK3 sites identified, T224 and S227, were crucial for necroptotic signaling, while the recently reported phosphorylation of activation loop residues, S164/T165 , negativRIPK3/−− HT29 cells  was set at 8000. All other settings in group or global parameters were left as default.Further analysis was performed using a custom pipeline developed in R (3.6.1), which utilizes the LFQ intensity values in the MaxQuant output file proteinGroups.txt. Proteins not found in at least 50% of the replicates in one group were removed. Missing values were imputed using a random normal distribution of values with the mean set at mean of the real distribution of values minus 1.8 s.d., and a s.d. of 0.3 times the s.d. of the distribution of the measured intensities. The probability of differential site modification expression between groups was calculated using the Limma R package (3.4.2).RIPK3 was deleted by CRISPR-Cas9 editing  or kinase-inactive D142N  mutant hMass spectrometry of FLAG eluates identified RIPK3 in all samples post-doxycycline induction, and the other core necrosome components, RIPK1 and MLKL, were also observed Fig. . As expeWhile the focus of our study was on the phosphorylation events occurring within RIPK3, mass spectrometry also identified several other proteins that were previously identified to interact with RIPK3 or involved in the TNF-induced necroptotic pathway Fig. . IKBKG  RIPK3 mutations were introduced in parallel as negative controls. Following 2.5 ng/mL doxycycline treatment to induce expression levels akin to those of endogenous RIPK3, the ability of each RIPK3 construct to reconstitute necroptotic cell death was examined by IncuCyte live cell imaging  or phosphomimetic (aspartate) residues, stably introduced each mutant into ing Fig. . Mutatioing Fig. . Upon neing Fig. , which wRIPK3/−− HT29 cells expressing S227A RIPK3 treated with TSI looked identical to untreated cells. Mutation of the adjacent T224 only decreased necroptotic cell death slightly, suggesting that pT224 is less crucial for RIPK3–MLKL interaction than pS227 (Fig. Phosphorylation of both human RIPK3 T224 and S227 contributes to RIPK3-mediated necroptosis, but intriguingly they are not essential for MLKL phosphorylation. The phosphomimetic S227D RIPK3 mutant also failed to reconstitute necroptotic cell death, as measured by SYTOX Green uptake Fig. , likely 227 Fig. . AlthougStrikingly, all single or double mutants of T224 and S227 retained the ability to phosphorylate MLKL Fig. , and theMLKL/−− HT29 cells, phosphorylation of RIPK3 S227 was not detectable under basal conditions (Fig. RIPK1/−− HT29 cells under basal conditions (Supplementary Fig. We sought to further evaluate the role of S227 phosphorylation in RIPK3-mediated signaling. In wild-type HT29 cells, RIPK3 pS227, but not MLKL pS358, was observed under basal conditions and during extrinsic apoptosis TS; Fig. . By compons Fig. , but wasRIPK3−/− HT29 cell reconstitution studies above (Fig. The observation of basally phosphorylated RIPK3 in HT29 cells was unexpected and raised the question whether this could arise from unforeseen behavior of the anti-pS227 RIPK3 antibody. In the ove Fig. . To elimove Fig. . Surprisove Fig. . Taken tThe necroptosis pathway relies on a cascade of post-translational modifications , which iSince the discovery of MLKL as the terminal effector of necroptosis, phosphorylation of RIPK3 S227 was known to be essential for MLKL interaction and necroptotic cell death  Fig. 5b5b. AlthoRecent studies of the endogenous human RIPK3:MLKL interaction support that idea that the recruitment of MLKL to RIPK3, or the necrosome, precedes the phosphorylation of MLKL , 37. A sIn keeping with the proposed pro-apoptotic role of pS164/pT165 , phosphoAll three clusters of phosphorylation sites that have been proposed to regulate RIPK3 functions, pT224/pS227, pS164/pT165 , and pT1In the present work, we defined the synergistic role of RIPK3 pT224 and pS227 in stably recruiting MLKL, which is independent from the phosphorylation of MLKL. We propose that RIPK3-mediated recruitment of MLKL to the necrosome is an upstream necroptosis checkpoint, which occurs prior to the phosphorylation of MLKL following a cell’s exposure to a necroptotic stimulus. Our data reveal additional RIPK3 autophosphorylation sites and sites phosphorylated by other kinases, in addition to RIPK3 interactors, which could aid future studies to uncover additional regulatory mechanisms of RIPK3-mediated pathways.Supplementary Figures 1-3Author checklist


The absence of photosynthetic oxygen evolution by PSII membranes without artificial electron acceptors, but in the presence of [CuL2]Br2, has shown that it is not able to act as a PSII electron acceptor. In the presence of artificial electron acceptors, [CuL2]Br2 inhibits photosynthetic oxygen evolution. [CuL2]Br2 also suppresses the photoinduced changes of the PSII chlorophyll fluorescence yield (FV) related to the photoreduction of the primary quinone electron acceptor, QA. The inhibition of both characteristic PSII reactions depends on [CuL2]Br2 concentration. At all studied concentrations of [CuL2]Br2, the decrease in the FM level occurs exclusively due to a decrease in Fv. [CuL2]Br2 causes neither changes in the F0 level nor the retardation of the photoinduced rise in FM, which characterizes the efficiency of the electron supply from the donor-side components to QA through the PSII reaction center (RC). Artificial electron donors  do not cancel the inhibitory effect of [CuL2]Br2. The dependences of the inhibitory efficiency of the studied reactions of PSII on [CuL2]Br2 complex concentration practically coincide. The inhibition constant Ki is about 16 µM, and logKi is 4.8. As [CuL2]Br2 does not change the aromatic amino acids’ intrinsic fluorescence of the PSII protein components, it can be proposed that [CuL2]Br2 has no significant effect on the native state of PSII proteins. The results obtained in the present study are compared to the literature data concerning the inhibitory effects of PSII Cu(II) aqua ions and Cu(II)-organic complexes.The effects of the novel [CuL Weeds, especially fast-growing and continuously emerging herbicide-resistant species, are one of the main factors limiting the yield of economically important crops. Advanced technology to increase yields through the creation of genetically modified plant species involves the simultaneous application of herbicides to kill weeds. Resistance to these herbicides has been created in cultivated plants through gene manipulation . HoweverPhotosynthesis ensures the growth and development of plants at the expense of the energy from the Sun, which is a unique characteristic of plant cells. Photosystem II (PSII), one of the main complexes of the photosynthetic apparatus (PA), splits water into electrons, protons, and oxygen . Among aAlong with mentioned inhibitors of the photosynthetic electron transport chain, and mainly PSII, several attempts have been made to develop new inhibitory organic complexes ,8,9,10, 2+) easily interact with various organic ligands, including ethylenediaminetetraacetic acid Br2) on photosystem II. To analyze the effects of this complex on the activity of PSII membranes isolated from spinach, we studied photosynthetic oxygen evolution by PSII membranes; the photoinduced changes of the PSII chlorophyll fluorescence yield (Fv) related to the photoreduction of the primary quinone electron acceptor, QA; as well as intrinsic fluorescence of aromatic amino acids (components of the PSII proteins); at different [CuL2]Br2 concentrations. The results obtained in the present study are compared to the literature data on the inhibitory effects of PSII Cu(II) aqua ions and Cu(II)-organic complexes.Various sites and effects of Cuorophyll . The Cu orophyll . The inh) buffer . By suppnto heat . The fachin PSII . Howeverhin PSII  showed tct to H+ . Cu inflA domain ,40. CuCl control . Furtherto P680+ . Cu2+  according to a method similar to the one described previously Br2 complex on the interaction between PSII and PSI through the electron transport chain was studied on thylakoids by a characteristic decrease in the yield of PSII chlorophyll fluorescence due to the oxidation of QA− upon the additional excitation of PSII by the so-called Light 1, which predominantly excites PSII, as in Br2 with PSII membrane proteins was investigated by monitoring the changes in the intrinsic fluorescence of the aromatic amino acids of these proteins according to the methods described previously in [2]Br2, were excited at a wavelength of 275 nm, with an excitation spectral slit width of 20 nm, and fluorescence emission was registered in a range from 290 nm to 420 nm, with an emission spectral slit width of 10 nm. Before the measurements, the samples were incubated in the presence of the studied complex in the dark for 3 min (or 21 min). The complex was added to the PSII membrane suspension in the DMSO solution. The DMSO concentration in all of the samples was the same as in the control (not more than 5%). The PSII–membrane concentration was equivalent to 10 µg mL−1 of chlorophyll.Possible changes in the state, structure, and the environment of protein(s) (including PSII proteins) as a result of the binding caused by an inhibitory agent can be detected by changes in the intrinsic fluorescence (mainly fluorescence intensity changes) of amino acids with aromatic ring side chains (tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe)), predominantly Trp and Tyr ,9,59. Thously in ,9,59. Th12H10N6S (mol. wt. 270.31): C, 53.32; H, 3.73; N, 31.09; S, 11.86. Found: C, 53.39; H, 3.79; N, 31.11; S, 11.89. IR : 3412, 3296, 1639, 1509, 1249. LC-MS: m/z = 270.31 (M+). 1H-NMR δ: 6.65 Ligand 4H-1,3,5-triazino benzothiazole-2-amine,4-(2-imidazole) was synthesized for the first time by a condensation reaction between aldehydes  and 2-guanidobenzimidazole or 2-benzothiazoleguanidine. A solution of 2-benzothiazoleguanidine (0.192 g) in methanol (25 mL) was treated with 2-imidazolcarboxylaldehyde (0.096 g) and two drops of piperidine. The mixture was stirred at 60 °C for 5 days. The resulting orange solid was separated by filtration, washed with methanol, and dried under a vacuum. Calculated for C2]Br2 were synthesized using an electrochemical method Br2, we tested [CuL2]Br2 as an artificial electron acceptor for PSII in a photosynthetic oxygen evolution reaction. However, in the presence of only [CuL2]Br2 (from 1 µM up to 30 µM) in the absence of other artificial electron acceptors (AEA), no oxygen evolution by PSII membranes was observed, as seen in Because the aqua ions of Cu(II) (1) can oxidize many inorganic and organic compounds (shown upper) (2), stimulate photosynthetic oxygen evolution at low concentrations ,62, and 2]Br2 (control), the PSII membranes generate oxygen under continuous illumination with saturating light at a rate of about 480 μmol O2 (mg Chl h)−1 (2]Br2 inhibits oxygen evolution in a concentration-dependent manner. At concentrations of 3 µM (2), 20 µM (3), 50 µM (4), and 100 µM the reaction rate is decreased by about 15%, 43%, 59%, and 69% versus the control (100%) (2]Br2 (not shown). The degree of PSII membrane inhibition does not depend on the duration of the incubation (5–20 min) in the presence of [CuL2]Br2.In the presence of AEA (DCBQ plus FeCy) without Br2 on PSII. The results of these studies are presented in The suppression of photosynthetic oxygen evolution is one of the most significant indications that the present agent is indeed an inhibitor of PSII functional activity. However, the results obtained in our study do not make it possible to judge whether (1) this agent acts on the donor or acceptor side or at the RC level, or (2) which intermediate of the PSII electron transport chain is affected by this agent, and (3) whether this agent acts as an electron transfer inhibitor or as an exogenous electron donor (acceptor) capable of effectively competing with native endogenous components and thus disrupting the normal course of PSII photochemical reactions. Information (a) on the localization of the inhibition site of this inhibitor in PSII, (b) on the number of sites of action and location of the main and/or additional site(s) of inhibition, and (c) on the possible mechanism of action of the inhibitor can be obtained using the photoinduced changes of the PSII chlorophyll fluorescence yield (FV) related to the photoreduction of the primary quinone electron acceptor, QA, in the PSII sub-chloroplast membrane particles in the absence of any additions (kinetics 1) and in the presence of [CuL2]Br2 at concentrations of 8 µM (kinetics 2), 30 µM (kinetics 3), and 100 µM (kinetics 4). In these experiments, we determined the values of F0 and FM as the maximum and minimum fluorescence yield of chlorophyll “a” in the dark-adapted samples. Correspondingly, FV calculated as FM–F0, the ratio of Fv/FM, recorded the possible changes in these parameters caused by the action of the inhibitor compared to the control. The control kinetics (measured in the absence of an effector) had an FV/FM ratio of 0.79 ± 0.02. This value indicates a rather high photochemical activity of PSII in the used PSII membranes. In the presence of [CuL2]Br2, a significant decrease in the FM level occurs (kinetics 2), and this decrease in the FM level is greater at higher concentrations of added [CuL2]Br2 (kinetics 3 and 4). It is very important to note here that at all of the studied concentrations of [CuL2]Br2, the decrease in the FM level occurs exclusively due to a decrease in FV; [CuL2]Br2 does not cause any changes in the F0 level (neither increase nor decrease) (kinetics 2–4). Therefore, to estimate [CuL2]Br2 inhibitory efficiency, we chose FV as a percentage relative to the control. The inhibition of PSII photoinduced FV is 66%, 48%, and 32% at [CuL2]Br2 concentrations of 8 µM (kinetics 2), 30 µM (kinetics 3), and 100 µM (kinetics 4), respectively. These data are in good agreement with the data obtained in experiments on photosynthetic oxygen evolution  both slowing down and acceleration of the dark relaxation of FM, reflecting that the dark reoxidation of the reduced QA occurs; (b) no retardation of the photoinduced rise in FM also occurs. Exogenous electron donors  do not remove the inhibitory effect of [CuL2]Br2 on FV, regardless of the sequence of addition of these reagents (not shown). Moreover, the degree of FV suppression by the same concentration of [CuL2]Br2 does not increase with increasing incubation time (3 and 21 min) in the presence of this inhibitor in the dark (not shown).volution . It is a2]Br2 concentration, measured as both photosynthetic oxygen evolution (black squares) and as light-induced changes in the PSII chlorophyll fluorescence yield (red circles). Both of the presented dependences were measured at a PSII membranes concentration equivalent to 20 µg mL–1 of chlorophyll. The dependence of the oxygen evolution inhibition almost completely coincides with that of the suppression of the photoinduced changes in the PSII chlorophyll fluorescence yields over the entire range of the investigated agent concentrations. Usually, a special indicator is used to quantitatively assess inhibitor potency. It is IC50  for unification)—the concentration of agent needed to inhibit the investigated activity to half that of the initial value (control) Br2 concentrations induce the suppression of half of the maximal studied activities (IC50), are about 30 µM. [CuL2]Br2 in concentrations of a little bit more than 0.1 µM only induces a very weak suppression of these PSII photochemical activities. An almost complete (more than 90% versus control) suppression of both reactions was observed at an agent concentration of 10 mM. It should be noted that all of the presented data were obtained at a concentration of PSII membrane of 20 µg mL−1 chlorophyll.As can be seen from the data presented in 50 depends on PSII membranes concentration, it cannot be used to compare the results obtained from different inhibitors or for the same inhibitors obtained by various scientific groups without specifying the target concentration for which that particular IC50 value was determined. In contrast to IC50, another parameter, Ki (the inhibition constant characterizing the intrinsic measure of binding affinity between the inhibitor and its target site), does not depend on the concentration of the PSII membranes. It is a known method for determining Ki based on the IC50 values data obtained for different concentrations of the target  [50 values for the suppression of light-induced oxygen evolution by [CuL2]Br2 for nine different concentrations of PSII membranes. We then plotted IC50 as a function of chlorophyll concentration as a line plot and extrapolated the resulting line to a zero chlorophyll concentration, as described previously [50 value that corresponds to a zero chlorophyll concentration is Ki (50 = Ki + 1/2 xt) given by [2]Br2-specific binding sites (xt) to the chlorophyll concentration. In the case of the PSII membrane concentrations used by us, for example, equal to 20 μg ml−1 or about 22 μM, we determined that xt equals 1, i.e., one [CuL2]Br2 molecule on one chlorophyll molecule.Because ICmbranes) . We alsoeviously . The reson is Ki . In our given by , it is p2]Br2 concentrations. It is known  that if A by PSI [2]Br2 affects the efficiency of light-induced oxidation by PSI of pre-reduced QA.A significant number of known inhibitors that affect PSII, act on its acceptor side, blocking the re-oxidation of reduced QA by PSI . In addiA by PSI . Given tA (QA−) from the FM-level to level F0 and to switch it on at a determined moment of actinic Light 1 (L1), predominantly exciting PSI (λ > 700 nm). The transmission spectrum of Light 1 is shown in A− re-oxidation is observed. For comparison, we used DCMU -1,1-dimethylurea) and DBMIB . DCMU and DBMIB are available, well studied, and the most often and ubiquitously used in scientific research inhibitors. The mechanisms of the inhibitory actions of these agents are well studied. DCMU blocks QA−, the oxidation by plastoquinone (PQ) from the membrane pool by binding instead of PQ with the QB-plastoquinone-herbicide-binding protein (QB-site of D1-protein) of the PSII reaction center Br2 affects the structure of the PSII proteins by comparing the parameters of the AAA intrinsic fluorescence in the suspension of the PSII membranes without and with [CuL2]Br2. The results of these studies are presented in Three amino acids with aromatic circular side chains—phenylalanine, tyrosine, and especially tryptophan—are sensitive to the local environment in the proteins, and these AAA undergo changes in the wavelength and/or intensity of their intrinsic fluorescence upon any impact on the protein . One of 2]Br2 at various times . Upon excitation with light at a wavelength of 275 nm, a broad band was observed in the emission spectrum with a broad maximum at 330 nm and a shoulder at 350 nm. This is the emission of the intrinsic fluorescence of tyrosine, tryptophan, and phenylalanine [2]Br2 has any effect on PSII proteins, then we could expect any changes in the intensity and/or any shift in the maxima of this fluorescence if the native configuration of the PSII proteins will be disturbed due to the action of [CuL2]Br2 or as a result of binding to the PSII proteins of the [CuL2]Br2 complex or the Cu2+ ions released from [CuL2]Br2. It is known that, in parallel with a decrease in the intensity of the intrinsic fluorescence of AAA in the protein, a shift in the F maximum is also possible. This indicates a change in the fluorophore microenvironment as a result of the effector impact Br2 at a concentration of 30 μM), the intensity of the intrinsic fluorescent emission of PSII proteins did not decrease in the presence of [CuL2]Br2 at any of the indicated wavelengths .However, in all of the cases , obviously, is not able to act as an exogenous PSII electron acceptor. These results could be taken as an indication that the copper cation in the complex is sufficiently strongly bound to the ligand, and the charge on the copper cation is balanced by a total charge on the ligand sufficient to prevent the possible interaction of the copper cation with other components of the environment as electron acceptor. The previously observed stimulation of the photosynthetic oxygen evolution by the Cu2+ at low concentrations Br2 is present in the measurement medium; it may indicate that [CuL2]Br2 inhibits the functional activity of some PSII electron transport chain components. However, it can also be assumed that this observed effect is the result of a direct chemical interaction between AEA-s and [CuL2]Br2  leading to the depletion of AEA-s concentrations and, as a consequence, to the decrease in the rate of oxygen evolution. It can be assumed that such an AEA in the used pair of AEA-s can be each of these agents or one of them: DCBQ or FeCN. If we compare the potentials of substances oxidized by the Cu(II) cation: ascorbate Em,7 = +60 mV Br2. However, the following experimental facts testify against this assumption: (1) The absence of changes in the absorption spectra of both FeCN and [CuL2]Br2 upon mixing and co-incubation for a sufficient time under the conditions of measuring the photosynthetic oxygen evolution rate but in the absence of the PSII membranes (not shown); (2) [CuL2]Br2 does not have any absorption bands in the red region of the spectrum (λ > 650 nm) (not shown). Therefore, a [CuL2]Br2 concentration-dependent decrease in the rate of the photosynthetic oxygen evolution is not result of the actinic light screening by [CuL2]Br2. The hypothesis that the decrease in the photosynthetic oxygen evolution by PSII membranes is a consequence of the direct interaction of [CuL2]Br2 with DCBQ or FeCN could be tested by increasing the concentration of FeCN and/or DCBQ. If the inhibition efficiency did not change, then this would testify against the above proposal. However, the concentration of FeCN is already quite high (1 mM), above which it is quite possible to expect non-specific effects of FeCN on the PSII-membranes. On the other hand, it is not recommended to use DCBQ at concentrations above 250–300 µM. Based on the experimental data, it is proposed that DCBQ taking an electron from the reduced species in PSII may then donate it to P680+˙ or TyrZ˙, while ferricyanide prevents this process through the oxidation of the reduced DCBQ [2 mg Chl−1 h−l) in the presence of DCBQ (without FeCN). It can be assumed that [CuL2]Br2 acts as an exogenous electron donor capable of competing with the endogenous OEC for positive equivalents from RC and, therefore, for the ability to supply electrons for the reduction of the DCBQ + FeCN pair, not directly as a result of a direct chemical reaction, but through the components of RC. This could be manifested as a decrease in the rate of light-induced oxygen evolution with an increase in the concentration of [CuL2]Br2. However, the above indirect data on the redox properties of Cu2+ aqua ions and the effect of organic ligands on them makes this assumption unlikely. The fact that the degree of inhibition of the photosynthetic oxygen evolution rate by PSII membranes does not depend on the time of incubation in the presence of [CuL2]Br2 in the dark  indicates that [CuL2]Br2 sufficiently reaches the target quickly, apparently localized in the hydrophobic region of the PSII pigment–protein complex. This assumption is also supported by the fact that [CuL2]Br2 is very poorly soluble in an aqueous media and, therefore, a stock solution of [CuL2]Br2 was prepared in DMSO. Based on the above reasoning, we are inclined to assume that the decrease in the photosynthetic oxygen evolution rate by PSII membranes observed in the presence of [CuL2]Br2 is the result of inhibition by [CuL2]Br2 of the functional activity of some component of the PSII electron transport chain. Earlier Br2 acts as an exogenous electron acceptor that removes an electron from QA more efficiently than the OEC supplies electrons to QA and therefore photoaccumulation of the reduced QA does not occur, and it is revealed as a decrease in FM. (c) [CuL2]Br2 is a quencher of the excited states of chlorophyll, and this manifests itself as the quenching of chlorophyll fluorescence. Let us consider these assumptions in reverse order.The PSII maximum quantum photochemical yield of the dark-adapted samples φPo = Fv/F2]Br2 only selectively reduces the intensity of the variable chlorophyll fluorescence (Fv) and does not affect the level of F0 , the assumption that [CuL2]Br2 acts as a fluorescence quencher seems unlikely.(d) [CuLel of F0 . Taking 2]Br2 acted as a very efficient exogenous electron acceptor, then (1) the dark decay of FV reflecting the rate of reoxidation of reduced QA could be greatly accelerated; (2) the photoinduced growth rate of FM could be decreased; (3) we could observe light-induced oxygen evolution in the absence of exogenous electron acceptors; (4) there could be spectral changes in the absorption spectrum of [CuL2]Br2, indicating the formation of the reduced form of this agent. However, none of the abovementioned occurs (2]Br2 does not act as an electron acceptor.(c) If [CuLd occurs . So [CuL2]Br2 is incorporated somewhere on the donor side or between its components and blocks and/or slows down normal electron donation and/or acts as an exogenous “poor” electron donor to the RC, unable to provide efficient photoaccumulation of reduced QA. However, the lack of slowdown in the rate of FM rise in the presence of [CuL2]Br2  is the evidence against the fact that the donor side is the site of the inhibitory action of this agent. In addition, the indirect data on the redox properties of [CuL2]Br2 discussed above also testify against this assumption.(b) It could be assumed that [CuL2]Br2 most likely is the components of the PSII reaction center. This assumption is quite convincingly confirmed by all the considerations discussed above, which reject other sites of action of this agent on PSII. In addition, as it was considered in the introduction, the most likely and the most experimentally substantiated main site of action among several auxiliary sites of action of both Cu2+ aqua ions and organometallic complexes based on Cu2+ seems to be components of the PSII reaction center.(a) Thus, the main site of the inhibitory effect of Br2 molecule on one chlorophyll molecule, or 220–250 inhibitor molecules per one PSII reaction, are different from the data. DCMU: 1 DCMU molecule per around 300 molecules of chlorophyll [2+ aqua ions (as CuCl2) or DCMU in the presence of 0.5 mM DCBQ as an artificial electron acceptor obtained by Yruela et al. in 1992 [2]Br2 binding site, the data that [CuL2]Br2 independently binds with only one binding site per acceptor molecule are a similar conclusion that was drawn for the inhibitor, 4.6-dinitro-o-cresol Br2 on the interaction of PSI with PSII (2]Br2 effect is no more than 5–10% of the DCMU effect. Comparing the effects of [CuL2]Br2 on FM (50%) and on the interaction of PSI with PSII (see above), it is reasonable to propose that the latter effect is probably additional and not main. The existence of additional sites (effects) to the main known that is assumed and experimentally shown for many inhibitors of photosynthesis Br2 compared to the control  indicates that (1) neither the whole [CuL2]Br2 complex nor the copper ion that could be released from this complex had no significant effect on the AAA PSII proteins native state; (2) and, therefore, there evidently is no release of the copper ion from the [CuL2]Br2 complex. Previously, it was shown that a number of water-soluble organometallic complexes based on Cu(II) effectively quench the intrinsic fluorescence of AAA of PSII proteins. Based on these and other data, it was proved that copper ions are released from these complexes and bind to tryptophan and tyrosine, thus inhibiting the functioning of the components of the PSII donor side [The absence of any change in the AAA fluorescence spectra in the presence of [CuLnor side . These c2]Br2 is a component of the PSII reaction centre. These data are consistent with the fact that Cu(II) aqua ions also act on PSII RC components [2]Br2 molecule can penetrate into RC components located in a relatively densely packed and hydrophobic RC region. However, on the one hand, [CuL2]Br2 itself is a highly hydrophobic compound (see above), and, on the other hand, it has been theoretically substantiated and experimentally shown that the RC as a whole and its individual protein components undergo numerous conformational changes in the process of functioning, as well as at different reversible and irreversible impacts [2]Br2 inhibition seems to be quite realistic. The data on the absence of quenching the intrinsic fluorescence of AAA by [CuL2]Br2 do not in the least contradict the assumption that the site of action of [CuL2]Br2 is RC. There are circular dichroism data that indicate that even in the case of a significant quenching of AAA intrinsic fluorescence by Cu2+ aqua ions due to their binding to a red fluorescent protein (DsRed) or to its derivatives, no effect of the Cu2+-binding on the secondary structure or on the conformation of these proteins was revealed Br2) PSII, (1) several different interesting effects were identified and studied at the level of different PSII regions. (2) The assumption was confirmed that the presence of a ligand significantly changes the possible site and mechanism of the inhibitory action of copper ions. The experimental data at the time of writing this article can be explained by the fact that a possible mechanism of inhibition could be some conformational changes in the structure of the reaction center due to its interaction with the inhibitory agent, especially during photoaccumulation. To elucidate the exact mechanism and site of the inhibitory action of this agent, it is planned to significantly expand the list of research methods and the types of biological samples and apply the method of stabilizing the structure of the reaction center. The results obtained can be used in the development of methods for the guaranteed delivery of the active element to the site of its main activity in the organic complex.According to the purpose of the work, to investigate the possible effects of a new organometallic complex, (CuL


Drosophila melanogaster has become a focal system for investigations of rapidly evolving genital morphology as well as the development and functions of insect reproductive structures. To follow up on a previous paper outlining unifying terminology for the structures of the male terminalia in this species, we offer here a detailed description of the female terminalia of D. melanogaster. Informative diagrams and micrographs are presented to provide a comprehensive overview of the external and internal reproductive structures of females. We propose a collection of terms and definitions to standardize the terminology associated with the female terminalia in D. melanogaster and we provide a correspondence table with the terms previously used. Unifying terminology for both males and females in this species will help to facilitate communication between various disciplines, as well as aid in synthesizing research across publications within a discipline that has historically focused principally on male features. Our efforts to refine and standardize the terminology should expand the utility of this important model system for addressing questions related to the development and evolution of animal genitalia, and morphology in general.The model organism  Animal terminalia  have a long history of being used for taxonomic and phylogenetic purposes, as well as being studied in the context of functional morphology and morphological evolution. This is because these structures possess a remarkable level of anatomical diversity, making them excellent morphological features for distinguishing species as well as understanding mechanisms of rapid morphological change . Past inDrosophila genus have become an important study system to address research questions in ecology, behaviour, evolution, development and taxonomy. A survey of the egg-laying apparatus of Hawaiian drosophilids for example revealed that ovipositor form, and especially length and patterns of sensory structures, differ between species and strongly correlate with adaptations to different oviposition substrates  on a multi-well glass slide.Reproductive tracts of the different treatments were mounted with Antifade media  on a mulDsRed expression (magenta) for an enhancer-reporter construct containing 301 bp of sequence between the transcription start site of CG32833 and a distal transcription start site of twist . Note that this intergenic sequence is also upstream of the transcription start site of miR-4939 (transcribed in the same direction as CG32833) and of the transcription start site of long non-coding RNA gene CR42742 (transcribed in the same direction as twist). It is not known to which gene’s expression pattern the reporter corresponds. The 301-bp fragment was amplified by PCR with primers respectively containing a KpnI site and an XhoI site, for cloning into the KpnI and XhoI sites in the polylinker of pRed H-Stinger  for 24–48 hours at 40°C. Before imaging, the samples were washed two times for ten minutes each in fresh 200 proof ethanol. Micro-CT was done with a Zeiss Xradia micro XCT-400 at X20 magnification and data processing was done using AVIzO and Fiji .