The widespread nature and economic importance of Infectious bronchitis virus (IBV) and interactions between IBV and the host immune response remain poorly understood. Understanding the mechanism of virus recognition via innate immunity can help resist IBV invasion. Retinoic acid-induced gene I-like receptor (RLRs) recognize virus RNA in virus infection, and LGP2 is a member of RLRs. According to the current studies, LGP2 exhibited certain inhibition in the virus, and there is a lack of investigation for chicken’s LGP2. It is important to figure out the role of chLGP2 in host immune recognition of IBV. Our results showed that chLGP2 inhibited the proliferation of IBV Beaudette in cells. Also, chLGP2 can identify and combine with IBV RNA. The domains of chLGP2 were separately expressed and inspired by related literature, and the chLGP2 K30A mutant was constructed. Our results suggested its structural integrity and the adenosine triphosphatase (ATPase) activity are critical for IBV inhibiting activity. chTRBP was selected after CO-IP and Mass spectrometry test. We found chTRBP and chLGP2 are the interacting partners and promote mutual expression. Our study showed that chTRBP could also suppress IBV infections via chLGP2, which provided a basis for future innate immunity research for IBV. Infectious bronchitis virus (IBV) causing infectious bronchitis (IB) is a highly contagious disease in poultry. Infectious bronchitis is more common in chicks causing bronchial rales, sneezing, and coughing, while some IBV serotypes also lead to kidney or fallopian tube diseases. Infectious bronchitis virus infection significantly deteriorates the quality and quantity of the poultry industry causing huge economic losses every year. IBV is a single positive-stranded non-segmented enveloped RNA virus that belongs to the family of Coronaviruses and is capable of infecting various mammals and birds. Based on serotype and genome, coronaviruses can be divided into four subgenres, namely alpha, beta, gamma, and delta coronavirus . InfectiCytoplasmic RLRs are the key recognition receptors for viral infections. retinoic acid-induced gene I-like receptor family (RLRs) recognize the RNA viruses and then mediate the production of type 1 IFN through signal transduction . LGP2, Rin vivo, and the LGP2 regulates this process in vivo (LGP2 (Laboratory of genetics and physiology 2) is a key part of the RLRs that shares high homology with MDA5 and RIG-I . In lack in vivo . Thus it in vivo , chLGP2 TRBP helps RNA silencing under siRNAs/miRNAs affect. TRBP is a double-stranded RNA binding protein family member, including three dsRNA binding domains. The first two are responsible for RNA binding and the C-terminal dsRNA binding domain for combining Dicer, PKR (protein kinase R), PACT (PKR associated activator). TRBP (Trans-activation response RNA-binding protein), Dicer, and Ago2 together constitute RISC (RNA-induced silencing complex) . StudiesTherefore, this study used an IBV model to analyze chLGP2 immunomodulatory roles in IBV infection, and we found that chLGP2 could inhibit IBV. Moreover, we found that chTRBP could interact with chLGP2. Thus chTRBP may participate in inhibiting IBV. Our results have certain reference significance for the future innate immunity research and prevention and control of IBV.50 test; the cells and the cell supernatant were collected at 3 h, 6 h, 12 h, 24 h, 36 h, 48 h post-infection. In both cell lines, the virus titer of the control group was significantly higher than that of the chLGP2 overexpression . Also, after IBV M41 infection, chLGP2 had no significant effect on IFN-β expression in DF-1 cells (P > 0.05), suggesting that chLGP2 maybe not be involved in the inhibition of IBV M41 strain in the DF-1 cells  .Results showed that overexpression of the chLGP2 helicase domain did not significantly reduce the replication of the IBV Beaudette strain, while the over-expression of chLGP2 CTD showed a certain inhibitory effect . Meanwhith and 89th bases of the chLGP2 open reading frame were double mutated to GCA (K) and AAA (A) to generate chLGP2 but lacked ATPase activity.The ATPase activity of LGP2 plays an important role in its function involving K30 residue. Accordingly, the 88The qRT-PCR results showed that overexpression of chLGP2 K30A inhibited the replication of IBV. However, the effect was significantly lower than wild-type . FurtherBased on the results above, we believe that chLGP2 functions differently and might involve other factors. The DF-1 and HD11 cells were plated on 6-well cell plates and transfected with chLGP2 plasmid (1 μg/well). After 24 h of IBV Beaudette strain infection, the cell lysates were collected and added with 40 μL anti-Flag affinity gel magnetic beads to analyze protein mass spectrometry (MS).The MS results showed that no IBV protein was bound to chLGP2. A total of sixty-five proteins bind to chLGP2, seventeen of which are shared in both cells, like calreticulin, TRBP, endoplasmic reticulum chaperone protein Bip , and stuFlag-tagged chLGP2 plasmid and HA-tagged chTRBP plasmid were co-transfected into the HD11 cells to verify the specific binding between chLGP2 and chTRBP. The cells were collected 36 h after co-transfection. WB analysis revealed that chLGP2 and chTRBP showed specifical binding in HD11 cells . So maybchTRBP, chLGP2, or chTRBP + chLGP2 were overexpressed in HD11 cells, and then the cells were infected with IBV to examine the relationship between chTRBP and IBV infection. The cells were collected at 3 h and 36 h post-infection. IBV replication and the expression of related anti-IBV immune genes were detected by qRT-PCR. As shown in Infectious bronchitis virus (IBV) is an acute and highly infectious chicken upper respiratory disease that causes bronchitis , leadingIn this study, chLGP2 was overexpressed in HD11 and DF-1 cells, and then the cells were infected with IBV Beaudette or M41 strain. The results showed that chLGP2 significantly inhibited the proliferation of IBV Beaudette. However, in DF-1 cells, chLGP2 had no significant effect on the replication of the IBV M41 strain, which may be due to the low replication of the IBV M41 strain in DF-1 cells, which may not be enough to activate the antiviral effect of chLGP2. There could be a unique immune escape in the cell process, which antagonizes the function of chLGP2. Also, the production of intracellular IFN-β showed a significant difference, indicating a different antiviral mechanism of chLGP2.The protein structure plays a decisive role in its function. chLGP2 is composed of a helicase domain and a CTD; the helicase domain has ATPase activity that binds to dsRNA. Studies showed that the combination of Paramyxovirus V protein and LGP2 inhibits the activation of MDA5 via LGP2, directly inhibiting the MDA5 mediated antiviral signal reducing the production of IFN and virus replication , 2014. CATP is a universal energy carrier in cellular activities and an important basis for many protein functions . Hei andSeveral studies analyzed the proteins that can specifically bind to LGP2 in the process of viral infection to understand the mechanism of LGP2. LGP2 can interact with endonuclease DICER1, PKR, PACT, NF-κB inhibitor (NKPF), and 5′-3′endo- nuclease 2 (XRN2). The interaction between LGP2 and PACT plays an important role in regulating RLRs signaling . Since cAmong the many interacting protein partners, chTRBP was selected for further investigation. Tomoko Takahashi et al. found that the helicase domain of LGP2 specifically binds to the dsRNA binding domain of TRBP to inhibit its pre-miRNAs recruitment function, thereby inhibiting the maturation of miRNAs . AnotherChicken macrophage cell line HD11 was presented by Professor Jiao Xin’an from Yangzhou University and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (HyClone), supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin-streptomycin (DMEM growth medium). Chicken fibroblast cell line DF-1 was from our laboratory and maintained in DMEM (HyClone), supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (DMEM growth medium).Both IBV Beaudette strain and IBV M41 strain (standard strain) were from our laboratory. For WB and immunofluorescence assays, the Flag-tag mouse monoclonal antibody (Beyotime Biotechnology), HRP-labeled goat anti-mouse IgG(H + L) (Beyotime Biotechnology), GAPDH Mouse Monoclonal Antibody (Beyotime Biotechnology), and Alexa Fluor 350 labeled goat anti-rabbit IgG (H + L) (Beyotime Biotechnology) were used as primary and second antibodies, respectively.HQ845773) was fused with Flag tag (GATTACAAGGAT GACGACGATAAG) and cloned between the EcoRI and BamHI restriction sites of the pcDNA3.1 vector. The open reading frame of chLGP2 is 2025 bp, encoding 674 amino acids. LGP2 comprises a helicase domain and a C-terminal regulatory domain connected by pincer. A Flag tag and a stop codon were added to the C-terminus of the helicase (1482 bp), and a start codon and a Flag tag were added to the N-terminus of the regulatory domain (612 bp) at the C-terminus. These were cloned using EcoRI and BamHI in the pcDNA3.1 vector. The open reading frame sequence of the chTRBP gene (gene accession number: XM_015300349.2) was fused with a HA tag (TACCCATACGATGTTCCAGATTACGCT) at the C terminus and cloned using EcoRI and BamHI. The above-designed cDNA sequences and plasmids were sent to Beijing Kinco Xinye Biotechnology Co., Ltd. for full gene synthesis. chLGP2 plasmids (500 ng/well and 1 μg/well) were transfected into HD11 cells and DF-1 cells using Lipofectamine™8000 Transfection Reagent (Beyotime Biotechnology); the empty plasmid (500 ng/well) was used as a negative control (The C-terminus of the open reading frame sequence of the chLGP2 gene (NCBI accession number  control .NM_001024836), F: GCTCTC ACCACCACCTTCTC and R: GCTTGCTTCT TGTCCTTGCT; IBV N (Accession no.AY851295), F: GAA GAAAACCAGTCCCAGA and R: TTACCAGCAAC CCA CA; chicken GAPDH (Accession no.NM_204305), F: CAT CACAGCCACA CAGAAG and R: GGTCAGGTCAACA ACAGAGA; chicken PKR (Accession no.NM_204487) F: CCTCTGCTGGCCTTACTGTCA and R: AAGAGAGGC AGA AGGAATAATTTGCC; chLGP2 (Accession no.HQ845773.1) F: AGCTCCACG GGTACCAACT and R: AGCTCCA CGGGTACCAACT; chTRBP (Accession no.XM_015300349.2) F: TGCAGGAGTATGGCACACGCAT and R: CGACGG TGACACGGAAGGTGAA; chicken BCL-2 (Accession no.NM_205339.2) F: TGT TTCTCAAACCAGACACCAA and R: CAGTAGGCACCTGTGAGATCG; chicken FasL (Accession no.AJ890143) F: GGCATTCAGTACCGTGACCA and R: CCG GAAGAGCACATTGGAGT; chicken IL-1β (Accession no.Y15006.1) F: CTG GGCATCAAGGGCTACAA and R: CGGTAGAAGATGAAGCGGGT; chicken Caspase3 (Accession no.NM_204725.1) F: TACCGGACTGTCATCTCGTTCAGG and R: ACTGCTTCGCTTGCTGTGATCTTC; chicken Caspase8 (Accession no.AY057939.2) F: GGAAGCAGTGCCAGAA CTCAGAAG and R: TTGTTGTG GTCCATGCACCGATAG; chicken Caspase9 (Accession no.BG711928.1) F: CCGAA G GAGCAAGCACGACAG and R: CATCTAGCATGTCAGC CAGGTCAC.Total RNA was extracted from cells using Trizol reagent (Invitrogen) and reverse-transcribed using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) following the manufacturer’s instructions. cDNA samples were subjected to polymerase chain reaction (PCR) amplification and electrophoresis to detect chLGP2 expression. The quantification of IFN-β and IBV N gene expression was quantified using SYBR green-based qRT-PCR on a CFX96 system . Primers used were as follows: chicken IFN-β  synthesized by Beijing Kinco Xinye Biotechnology Co., Ltd. Mut Express MultiS Fast Mutagenesis Kit  V2 was used to introduce continuous double base mutations in chLGP2 gene following the kit instructions. The sequence of the mutant plasmid was validated by Beijing Kinco New Industry Co. Ltd.50 value of IBV in the supernatant was estimated using the Reed-Muench method.The cells were spread in a 96-well plate and cultured to 80% confluency. The cells were infected with IBV, and the cell supernatant was collected 36 h after infection, which was 10-fold serially diluted with Opti-DMEM, 8 replicate holes per gradient. Also. The cells with the same Opti-DMEM medium were set as a negative control. After 48 h, the cytopathic condition of each dilution was observed, and the number of lesions in each gradient was counted. The TCIDThe cells were harvested and lysed in RIPA lysis buffer . An appropriate protein sample was mixed with the loading buffer and heated at 100°C for 15 min. The heat-denatured samples were resolved using pre-configured sodium dodecyl sulfonate-Poly- acrylamide gel electrophoresis (SDS-PAGE) gel at 120 V. The protein bands from the SDS-PAGE gel were transferred to the methanol-activated PVDF at 90 V for 1.5 h. After the transfer, the PVDF membrane was soaked in 5% skimmed milk in PBST  at room temperature (RT) for 2–3 h or overnight. The blocking solution was removed, and the membrane was washed with PBST 3 times, 5 min each time. Then, the membrane was incubated with Flag-tag mouse monoclonal antibody (1:1000) and GAPDH mouse monoclonal antibody (1:1000) at RT for 2–3 h, followed by 3 times washing with TBST as earlier. Next, the membrane was incubated with HRP-labeled goat anti-mouse IgG (H + L) antibody (1:1000) at RT for 1–2 h. The membrane was washed 3 times with TBST for 5 min each, and ECL and DAB were used for color rendering. The image was developed using a ChemiDoc Touch Imaging System (Bio-rad).An indirect immunofluorescence experiment was performed after cell culture for 24 h. The two 6-well cell culture plates with HD11 and DF-1 cells were removed from the cell incubator, and the cell supernatant was discarded. The plates were gently rinsed with PBS 3 times, 1 min each time. Next, 1 mL of 4% formaldehyde fixing solution was added to each well at 4°C for 15 min. Then, the fixative solution was removed, and the wells were washed with PBS 3 times, 1 min each time. 1 mL of immunostaining blocking solution was added to each well at 4°C. Subsequently, the immunostaining blocking solution was removed, and again 3 times washing was done with PBS. 1 mL of diluted Flag monoclonal antibody solution (500-fold dilution) was to each well for incubation at 37°C for 2 h. After rising with PBS, 1 mL of secondary antibody (diluted 500 times) was added to each well in the dark for 2 h incubation. After PBS washing, nuclear staining solution DAPI was added in the dark for 20 min incubation at 37°C at RT. The DAPI dye solution was discarded, and the wells were rinsed with PBS for observation under a fluorescence microscope (Zeiss CELL Observer SD).HD11 cells were spread in 6-well cell plates, and the cells were transfected with chLGP2, chLGP2 helicase, chLGP2 CTD, and chLGP2 K30A mutant (1 μg/well) at cell density 70–80%. Cells were infected with the IBV Beaudette strain after 24 h. Add protease inhibitors and RNase inhibitors to the cell lysate, and add the prepared lysate to the cells (200 μL/well). After 30 min of ice bath lysis, the cells in each well were collected in a 1.5 ml centrifuge tube. Centrifuge at 12000 rpm for 5 min at 4°C, take the supernatant, place it in a new 1.5 mL centrifuge tube, and place it in an ice-water bath. Pipette 20 μL of Anti-Flag gel suspension into the centrifuge tube with lysis buffer, incubate on ice, and shake for 2 h. Centrifuge at 6000 rpm for 30 s at 4°C, discard the supernatant, add 0.5 mL TBS and wash 3 times, place on a shaker in an ice bath for 5 min, repeat washing three times. Add 20 μL proteinase K to each tube, incubate at 42°C for 2 h, extract RNA.DF-1 and HD11 cells were spread in 6-well cell plates, and the cells were transfected with chLGP2 plasmid (1 μg/well) at cell density 70–80%. After 24 h, the cells were infected with the IBV Beaudette strain. The cell mixture was lysed and centrifuged at 6000 rpm for 3 min at 4°C. The obtained supernatant was transferred to a new centrifuge tube and added with 40 μL anti-Flag affinity gel magnetic beads. The mixture was incubated at 4°C overnight on a shaker. The next day, after careful washing with PBS 2–3 times, washed beads samples were collected and analyzed for the protein profile that interacted with chLGP2.t-test. P values < 0.05 were considered statistically significant.Data were analyzed with Graphpad Prism 9, and all values are expressed as mean ± standard deviation (SDM). Statistical analyses were conducted using Student’s This article analyzes the role of chLGP2 in infected cells for the first time and found that chLGP2 can recognize and bind IBV RNA, and the structural integrity of chLGP2 and ATPase activity plays an important role in its inhibition. It is proved that chTRBP can combine with chLGP2 to inhibit the replication of IBV in cells. The molecular mechanism of chLGP2 inhibiting the proliferation of IBV is further analyzed. It is also the first study of the role of chTRBP protein in the process of IBV and avian virus infection.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.KW and PC performed the experimental and computational work, coordinated the research, and contributed to the writing. Other authors have given some help and suggestions during the experiment and the writing of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The Eu3+ luminescencespectroscopy of the doped samples and of Ca2EuSbO6 has been carefully investigated upon collection of the excitation/emissionspectra and luminescence decay curves of the main excited states.Surprisingly, apart from the dominant red emission from 5D0, all the doped samples show an uncommon blue and greenemission contribution from 5DJ . This is made possible thanksto both multiphonon and cross-relaxation mechanism inefficiencies.However, the emission from 5D3 is more efficientand the decay kinetics of the 5DJ  levels is slower in the caseof Y- and Lu-based doped samples. This evidence can find a possibleexplanation in the crystal chemistry of this family of double perovskites:our structural investigation suggests an uneven distribution of theEu3+ dopant ions in Ca2YSbO6 andCa2LuSbO6 hosts of the general A2BB′O6 formula. The luminescent center is mainlylocated in the A crystal site, and on average, the Eu–Eu distancesare longer than in the case of the Gd- and La-based matrix. Theselonger distances can further reduce the efficiency of the cross-relaxationmechanism and, consequently, the radiative transitions are more efficient.The slower depopulation of Eu3+ 5D2 and 5D1 levels in Ca2YSbO6 andCa2LuSbO6 hosts is reflected in the longer riseobserved in the 5D1 and 5D0 decay curves, respectively. Finally, in Ca2EuSbO6, the high Eu3+ concentration gives rise to anefficient cross-relaxation within the subset of the lanthanide ionsso that no emission from 5DJ  is possible and the 5D0 decay kinetics is faster than for the doped samples.Eu 3+ as a dopant ion in theseries of doubleperovskite Ca2LnSbO6 hosts shows a different occupation of the available A and B crystalsites; this strongly affects the luminescence behavior in the visiblespectral region.Eu Depending onthe nature and size of the other elements, the rare earth ions canoccupy the A-site or B-site. In the Ca2LnRuO6 (Ln = La–Lu) system,4 which crystallizesin the monoclinic P21/n space group, the Ca2+ and Ln3+ cations arepartially disordered in the A-site and B-site positions of the A2BB′O6 double perovskite, and the Ru(V) cationsare located at the B′-site; therefore, the general formulaof these compounds is (Cax2–Lnx)(Lnx1–Cax)RuO6. The abundance of Ln3+ located at the B-site varies with its cationic radius: the largerLn cations tend to occupy the A-site, whereas the smaller Ln cationstend to enter the B-site. Similar crystal chemistry is expected forantimonates with double perovskite materials and Ca2LnSbO6 formula. Although a systematic study on their crystal chemistryis still missing in the literature, two components of this family(Ca2LaSbO6 and Ca2YSbO6) have been effectively employed as hosts of luminescent trivalentlanthanide ions. In particular, Ca2LaSbO6, whichcan be obtained with Eu3+ up to 80% substituting La3+, has been considered a useful red phosphor.6 Another efficient red phosphor can be obtained by doping Ca2YSbO6 with Eu3+ ion. The codoping withBi3+ has been reported to enhance the intensity of thered emission.7 Considering the crystalchemistry of the host, Y3+ ions are supposed to occupyonly the centrosymmetric B site. Nevertheless, Eu3+ shouldbe located in a noncentrosymmetric crystal site, since the 5D0 → 7F2 band dominates theluminescence spectrum.8 Since Ca2+ is located in the A-site (C1 point symmetry),the authors reasonably assumed a Ca2+/Eu3+ substitutionand the presence of a charge compensation mechanism. Finally, Ca2YSbO6 is also an effective host for other luminescentions, such as trivalent Sm, Dy, Ho, and Er.9Rareearth double perovskite materials with the general formulaA2LnSbO6 family, we have found it interestingto undertake a structural study on Ca2LaSbO6, Ca2GdSbO6, Ca2LuSbO6, and Ca2YSbO6 doped with 1 mol % Eu3+, and neat Ca2EuSbO6, by means of X-ray diffraction.The effects of the different nature of the hosts on Eu3+ luminescence spectroscopy have been also discussed and some structuraldetails have been revisited. This study, focusing on the structural/spectroscopicrelationship, reveals the presence of unusual spectroscopic featuresof Eu3+ when introduced as an impurity in these antimonatehosts.Due to the lack of a comprehensive study on the crystal chemistryof the Ca22.13+-doped Ca2LnSbO6  and Ca2EuSbO6 were prepared by solid-state reaction in and airatmosphere. CaCO3 (>99%), Sb2O5 (99.995%),Ln2O3 , and Eu2O3 (99.99%) were thoroughly mixedand pressed into pellets under a pressure of 10 tons. The samplesunderwent two heat treatments: the first one at 600 °C for 6h to eliminate carbonates and the second one at 1400 °C for 24h with a slow cooldown of 3 °C/min. Intermediate grindings wereperformed to improve the homogeneity of the materials.Crystallinesamples of 1 mol % Eu2.2X-ray diffraction(XRD) patterns were measured with a Thermo ARL X’TRA powderdiffractometer, operating in the Bragg–Brentano geometry andequipped with a Cu-anode X-ray source ,using a Peltier Si(Li)-cooled solid-state detector. The patterns werecollected with a scan rate of 0.002°/s in the 18–120°2θ range. Polycrystalline antimonate samples were ground ina mortar and then put in a side-loading sample holder for data collection.10 The instrumental X-ray peak profile functions and the sample displacement(SHFT variable) were determined by Rietveld refinement of the diffractionpattern of the LaB6 powder standard reference material(NIST 660C).The General Structure Analysis System (GSAS) program was employedfor the Rietveld refinement calculations.3TeO6 determined in a study by Hottentot and Loopstra11 in which Sb has been located in the place ofTe and Ca, and Ln ions shared the two crystallographic positions ofCa.. The following structural refinement strategy has beenperformed: (i) refinement of the background functions (shifted Chebyschev),scale factor, and cell parameters; (ii) refinement of the occupationfactors (OFs) of Ca and Ln in the two available crystal sites; (iii)refinement of the fractional atomic coordinates for Ca/Ln in the 4e crystal site (site A); (iv) refinement of the fractionalatomic coordinates for the oxygen atoms; (v) refinement of the isotropicthermal parameter (Uiso) for Ca, Ln, andSb ions; (vi) refinement of the isotropic thermal parameter (Uiso) for oxygen atoms ;(vii) global refinement of all structural variables mentioned above.In each step of the Rietveld calculation, also the GU, GV, GW, LX,LY, and asym profile terms of the pseudo-Voigt profile function no.2, included in the GSAS program, were refined. Due to its very lowconcentration (1 mol %), the presence or absence of Eu3+ within the structural model is expected to not change, in a significantway, the result of the Rietveld refinements. For this reason, forthe sake of simplicity, the presence of Eu3+ is not consideredduring the structural calculation. Crystal data such as atomic fractionalcoordinates, OFs, and Uiso for 1% Eu3+-doped Ca2LnSbO6 and Ca2EuSbO6 are reported in the Supporting Information (Tables S1–S6), along with other relevantpowder diffraction data (see the Powder Diffraction Data section).The reference structural model exploited in theRietveld calculationwas the one pertaining to the isostructural perovskite-like Ca2.3Room-temperatureluminescence spectra and decay curves were measured with a Fluorolog3 (Horiba-Jobin Yvon) spectrofluorometer, equipped with a Xe lamp,a double excitation monochromator, a single emission monochromator(mod. HR320), and a photomultiplier in photon counting mode for thedetection of the emitted signal. All the spectra were corrected forthe spectral response of the setup.33.1In 2LnSbO6  areshown in The observed and fitted XRD patterns of doped CaInspection of this figure showsthat there is good agreement betweenthe observed and refined powder patterns. The refined lattice parametersand OFs are given in 2+ and Ln3+ cations are partially disordered in the A-site(C1 point symmetry) and B-site (iC point symmetry) positions of the A2BB′O6 double perovskite and the Ca/Ln distribution over thesetwo available crystal sites is strongly dependent on the nature ofthe Ln ion. When small Lu and Y are considered, the trivalent ionshows a strong preference for the site with coordination number (CN)6  is almostfully occupied by Ca2+  are not significantly affected by thenature of the trivalent ion and the Ca/Ln–O distances deviatefrom 2.55 Å only for Ln = La . This behavior is quite similar to the one observed forthe Ca2LnRuO6 (Ln = La–Lu) double perovskitefamily, already discussed above.4As expected, according to Vegard’slaw, the cell size increasesas the ionic radius of the trivalent lanthanide ions increases 1. Ca2+ ar (CN)6 2, site B by Ca2+ 2. Obviou Ln = La 3. On the2LaSbO6 host, while the La ion has been calculated to exclusivelyoccupy site A, by Yin et al.,6 we foundthe presence of a small percentage of La3+ (2%) in thecrystal site B. The most important discrepancy concerns the Ca2YSbO6 host. In the literature, Y3+ ionsare supposed to occupy only the centrosymmetric octahedral B site,7 while we detect the presence of this ion alsoin site A , this finding should have a significantimpact. In fact, according to our conclusions, we can assume thatEu3+ could occupy both crystal sites A and B without thenecessity of involving charge compensation mechanisms.We also point out some discrepancy with the crystal data presentin the current literature. In the case of the Ca3.23+-doped Ca2LnSbO6  and Ca2EuSbO6 are shown in Imax of the 7F0 → 5L6 transition.The normalized RT excitation spectraof 1% Eu3+ excitation peaks. Also, an O → Eu charge transfer (CT) band below300 nm is detected (not shown).All compounds show various intense EuImax of the 5D0 → 7F2 transition.Upon excitation at 394 nm, wehave obtained the luminescence emissionspectra shown in 3+ 5D0 excited state. A close inspectionof the peak around 580 nm  showssignificant differences between the different compounds  than for Ca2YSbO6 and Ca2LuSbO6 hosts . The Eu3+ ion, which is supposed to substitutefor the Ln3+ one, can be located in the two available crystalsites  the Eu3+ ion can occupy both available crystal sites in Ca2EuSbO6 , the similarity of the Ca(Ln)–Obond distances for each site suggests the presence of Eu in both availablepositions , the crystal site B appears too small to accommodate Eu3+ in the case of Ca2YSbO6 and Ca2LuSbO6 matrices. In fact, the sum of the ionic radii ofEu and O  is significantly higher than the aforementionedCa(Ln)–O distances. In these hosts, the probable predominantoccupation of site A by the Eu3+ ion is supported by thesmaller FWHM of the 0-0 peak  induces the removal of the local inversion symmetryin the case of ions formally occupying centrosymmetric sites, froma crystallographic point of view.16 Therefore,also the Eu3+ emission from the cationic site B in Ca2LnSbO6 can occur through a forced electric dipolemechanism and, therefore, also the 5D0 → 7FJ  transition can be detected. The different luminescence emissionfeatures of Eu3+ . Nevertheless, in view of the complex crystal structureof the hosts and the structural disorder possibly inducing the breakingof the local inversion symmetry of B sites,16 other factors can contribute to determine the observed spectroscopicfeatures.In general, the results obtained uponpulsed excitation appearto be different for the doped samples with Ln = Lu and Y with respectto the ones with Ln = Gd and La. In parallel, the emission intensityfrom the ed hosts 7a. This 5D3, 5D2, 5D1 , and 5D0 (mainly in the red), the finalemission color can be tuned. The point 4 in the CIE diagram (2EuSbO6) canbe moved toward the green region (points 3 and 5) thanks to the presenceof a significant green component (the 5D1 → 7F1 band around 535 nm) in Ca2GdSbO6 and Ca2LaSbO6 hosts. A significantblue component  hasbeen observed for the Ca2LuSbO6 and Ca2YSbO6 hosts so that points 1 and 2 are closer to the bluespectral region (It is interesting to note that due to the differentcontributionsof the emission stemming from  diagram 11 (correl region 11.43+ (1 mol%)-doped Ca2LnSbO6 samples show unusual spectroscopic features,which are connected to the low-energy vibrational modes of the antimonatedouble perovskite and to the different site distribution of the luminescentEu3+ dopant ions. In particular, the color of the emittedlight can be tuned by simply acting on the nature of Ln ions in thehost. The typical red emission of Ca2EuSbO6 canbe slightly shifted toward the green and blue spectral regions when99% of Eu is replaced by La (or Gd) and by Lu (or Y), respectively.This is made possible thanks to the inefficient multiphonon relaxationprocess among 5DJ levels inthe doped samples. In this way, we observe emission in the 460–570nm region (from 5D1 and 5D2) in all the Eu3+-doped samples and emission in the 400–450nm region (from the 5D3 level) mainly in theEu3+-doped Ca2LuSbO6 and Ca2YSbO6 materials. In general, the emission from the 5DJ (J = 0–3)levels of Eu3+ is more efficient in the Y- and Lu-basedhosts and a possible explanation for this behavior can be found inthe crystal chemistry of these materials. The structural investigationon the host matrix suggests a different occupation of the cationicsites by the Eu3+ dopant, which should be preferentiallylocated in the A site when Ln = Y or Lu, while they can occupy bothavailable crystal sites (A and B) in the case of Ca2LaSbO6 and Ca2GdSbO6 hosts.Eu3+ ions (a nonradiativeenergy transfer pathway) is weakly active inall the doped samples, it is expected to be more efficient when theEu–Eu distances are shorter. This is the case of the La- andGd-based compounds, where the shortest possible Eu–Eu distanceis around 3.29 Å vs 3.90 Å in the caseof Ca2LuSbO6. As a consequence, the emissionintensity from the 5D3 level is significantlylower and the decay of the 5D0, 5D1, and 5D2 levels is faster forGd- and La-based hosts. Finally, as a consequence of the drastic increasein Eu3+ concentration, in the case of Ca2EuSbO6, an efficient cross-relaxation mechanism involving 5DJ levels takes place and no emissionfrom 5DJ levels  is possible. In addition, a fast migration of theenergy toward killer centers is responsible for a drastic decreasein 5D0 excited state lifetime.Althoughthe cross-relaxation mechanism involving EuThis contributionclearly shows how important is the detailed knowledgeof the host crystal chemistry and of the nonradiative mechanisms takingplace to control the luminescence features of an optical material.


The 2018 boreal summer in the Western North Pacific (WNP) is highlighted by 17 tropical cyclones (TC)—the highest record during the reported reliable years of TC observations. We contribute to the existing knowledge pool on this extreme TC frequency record by showing that the simultaneous highest recorded intensity of the WNP summer monsoon prompted the eastward extension of the monsoon trough and enhancement of tropical convective activities, which are both favorable for TC development. Such changes in the WNP summer monsoon environment led to the extreme TC frequency record during the 2018 boreal summer. Meanwhile, the highest record in TC frequency and the intensity of the WNP summer monsoon are both attributed with the combined increase in the anomalous westerlies originating from the cold tropical Indian Ocean sea surface temperature (SST) anomalies drawn towards the convective heat source that is associated with the warm central Pacific SST anomalies. Our results provide additional insights in characterizing above normal tropical cyclone and summer monsoon activities in the WNP in understanding seasonal predictable horizons in the WNP, and in support of disaster risk and impact reduction. There were 17 tropical cyclones (TC) that formed in the Western North Pacific (WNP) basin during the 2018 boreal summer —the highest recorded TC frequency since 1984 , but both reports differ in their choice of seasonal scaling. The first paper used July–November1 while the second report selected June–September2. Meanwhile, the other two papers focused on the WNP TC frequency but they also differ in their choice of seasonal scaling. The third paper selected June–November3 while the most recent report divided the WNP TC frequency into two seasons: JJA and September–October4. However, these choices of regional scaling and seasonal clustering do not totally account for the variability that is inherent to each basin and season. For example, we show that the WNP TC frequency in JJA has no significant correlation with the TC frequency in the Eastern North Pacific in the same season (r = 0.22)  to the existing knowledge pool in understanding the large-scale drivers behind above normal TC frequency and activity in the WNP.Questions were expectedly raised on what caused such unprecedented increased TC frequency in the WNP hence it is not surprising that a number of reports have already contributed to the existing knowledge pool6. When combined altogether, these anomalies characterize the highest TC frequency in JJA 2018.In JJA 2018, the WNP was highlighted by an anomalous low pressure region flanked by anomalous westerlies to its south and anomalous easterlies to its north, which converged in a zonally-elongated tropical convective activity, anomalous background cyclonic vorticity and wider WNP subtropical high p = 0.000)8 9, and Indian Ocean Basin Wide SST 11  and the Niño 4 index 12  plus there is no distinguishable anomalous SST dipole pattern between the western and eastern tropical Indian Ocean in JJA 2018 8 Fig. b, WNP su018 Fig. a.Figure 14. Furthermore, a positive IOBW is also found to force a surface divergence in the WNP emanating from an equatorial Kelvin wave that propagates into the WNP. A suppressed convective activity on top of an anomalous anticyclonic circulation and low-level divergence in the WNP are unfavorable environment for increased TC development in the WNP. Meanwhile, it is widely thought that warm tropical Pacific SSTs (e.g. Niño 4) induce increased surface fluxes, anomalous low-level westerlies and stronger vertical upward motion, which enhance pre-existing convective activities necessary for TC development17. Furthermore, a warm PMM contributes to an increased WNP TC frequency by prompting a weaker vertical wind shear and increased relative vorticity, primarily in the Southeastern portion of the WNP, which generally result to more TC development in the WNP19. The influence of WNPMI to TC frequency is discussed in the following section.It is reported that a positive IOBW contributes to the formation of an anticyclonic circulation in the WNP that suppresses convective activities particularly during July and Augustp = 0.062) and PMM  becomes marginally significant, and insignificant with the Niño 4 index , respectively. When the IOBW, Niño 4, and PMM are individually controlled, the partial correlations between the WNPMI and TC frequency remains significant at r = 0.50 (p = 0.003), r = 0.55 (p = 0.001) and r = 0.62 (p = 0.000), respectively, which suggest that the WNPMI controls the variability of TC frequency more than the IOBW, Niño 4, and PMM.We performed partial correlation analysis to test the influence of WNPMI, IOBW, Niño 4, and PMM to the WNP TC frequency Table . When thp = 0.000) and marginally significant with the PMM . When the PMM is controlled, the partial correlation between TC frequency and IOBW remains significant  and insignificant with Niño 4 index . Lastly, when the Niño 4 index is controlled the partial correlation between TC frequency and IOBW remains significant  and insignificant with the PMM (r = 0.27), which means that the IOBW has higher influence to TC frequency than the Niño 4 index and PMM, respectively. The higher partial correlation of both Niño 4 and IOBW is anchored on the fact that they have contrasting correlation signal with TC frequency hence when the influence of the other is controlled their respective zero-order correlation with TC frequency becomes higher.Moreover, when the IOBW is controlled, the partial correlation between TC frequency and Niño 4 becomes significant at r = 0.60 (p = 0.026) and not with PMM (r = − 0.16)  and is practically not correlated with the PMM (r = − 0.05). It is previously demonstrated that the PMM is more correlated with TC genesis at around 160°E and is not correlated with TC genesis westward of 160°E12. In JJA 2018, the mean TC genesis point is located around 140°E  has influenced the highest TC frequency in JJA 2018Both IOBW and the Niño 4 did not reach their extreme values nor are they unusually abnormal in JJA 2018  and 850 hPa wind anomalies in JJA 2018 and 1998, years with strong and weak WNPMI, and years with more and less TC frequency, respectively  than the Niño 4  (Table p = 0.000), and the partial correlation between the WNPMI and Niño 4 while controlling for IOBW is r = 0.58 (p = 0.001). The test of power of correlation between IOBW and Niño 4 suggests that there is 94.6% chance that the correlation coefficients are similar with each other and the risk of committing a Type II error while such similarity is false is only 5.4%. Therefore, both IOBW and Niño 4 have similarly influenced the WNPMI. We recognize that the WNP summer monsoon is also modulated by a variety of other factors such as the Intertropical Convergence Zone (ITCZ)21, WNP subtropical high, interhemispheric and regional thermal contrasts , but such matter is another subject for future investigation.One remaining open question is, between the IOBW and Niño 4, which has more influence to the WNPMI? The WNPMI is more significantly correlated with the IOBW , which is another intraseasonal mode of tropical convective activity in the Asian monsoon region, was also active when it was located in its Phases 2 and 8 (nearest to the Philippine Sea and WNP)24. These active tropical convective activities imply that the intraseasonal modes may have concurrently influenced the extreme TC record in JJA 2018 frequency. In addition, most TC genesis points are embedded in the daily OLR and 850 hPa zonal winds in JJA 2018  with highest WNP TC frequency and most intense WNPMI. The large-scale circulation patterns in JJA 2018 that prompted the recorded highest TC frequency during can be summarized as follows: a cool tropical Indian Ocean SST (negative IOBW) is associated with an anomalous anticyclonic circulation in the Indian Ocean, which generates low-level anomalous westerlies drawn towards the anomalous convective heat source in the central Pacific associated with warm central Pacific SST anomalies (positive Niño 4) Fig. . The strp = 0.012) and the combined TC frequency in the WNP and the Eastern North Pacific  in JJA than the WNP TC frequency alone  were considered in our analysis. Similar rules were applied for the counting of TC frequency in the Eastern North Pacific.The TC best track data are obtained from the International Best Track Archive for Climate Stewardship version 4 from 1984–201828. In the composite analysis, a year with more (less) TC frequency is defined as a year that is greater than or equal  to the 75th (25th) percentile range of the base period (1984–2018). Same definition of percentile rank is used for the strong and weak WNPMI. The years with more TCs include 1989, 1992, 1994, 1997, 2001, 2004, 2013, 2017, and 2018 while the years with less TCs are 1986, 1987, 1995, 1998, 1999, 2003, 2007, 2008, 2010, 2014, and 2015. Meanwhile, the years with strong WNPMI are 1985, 1986, 1990, 1994, 2001, 2002, 2004, 2012, and 2018 while the years with weak WNPMI are 1988, 1993, 1995, 1996, 1998, 2007, 2008, 2010, and 2017.We used the Japanese Reanalysis 55-year Project from 1984–2018 in our observational analysis7. The location of the monsoon trough is identified using the overturning 0 contour line of 850 hPa zonal wind in the WNP.Western North Pacific Monsoon Index—difference of zonal winds at 850-hPa between a southern region  and a northern region  10Indian Ocean Basin Wide SST—areal-averaged JJA SST at 20°S–20°N, 40–100°E11. We used the Niño 4 index as the ENSO flavor of our analysis because it is the most proximal classic Niño region to the WNP.Niño 4 index—areal-averaged JJA SST anomalies at 5°S–5°N, 160–210°E11Pacific Meridional Mode index—maximum covariance mode of meridional SST in the Pacific 8. The location of the WNP subtropical high index is identified using the 5880 gpm contour line.Western North Pacific subtropical high index—areal-averaged 850 hPa geopotential height at 15–25°N, 115–150°E The climate indices used in our analysis are defined as follows:The Pearson’s correlation is the selected measure of bivariate correlation and partial correlation in our study where their significance is tested using t-test with two-tailed distribution. The significance of difference is testing using student’s t-test. We applied 1,000 simulations in the student’s t-test using Monte Carlo Test to minimize the influence of uncertainty in the significance testing. The power for comparing two correlation coefficients was tested using Fisher-Z transformation.Supplementary Figures.


RGA1, a Cdc42-specific GTPase-activating protein (GAP), each lacking its C-terminal catalytic domain, that permit activation of the mating MAPKs under hyperosmotic conditions despite Hog1 being present. We show that Rga1 down-regulates Cdc42 within the high-osmolarity glycerol (HOG) pathway, but not the mating pathway. Because induction of mating pathway output via crosstalk from the HOG pathway takes significantly longer than induction of HOG pathway output, our findings suggest that, under normal conditions, Rga1 contributes to signal insulation by limiting availability of the GTP-bound Cdc42 pool generated by hypertonic stress. Thus, Rga1 action contributes to squelching crosstalk by imposing a type of “kinetic proofreading”. Although Rga1 is a Hog1 substrate in vitro, we eliminated the possibility that its direct Hog1-mediated phosphorylation is necessary for its function in vivo. Instead, we found first that, like its paralog Rga2, Rga1 is subject to inhibitory phosphorylation by the S. cerevisiae cyclin-dependent protein kinase 1 (Cdk1) ortholog Cdc28 and that hyperosmotic shock stimulates its dephosphorylation and thus Rga1 activation. Second, we found that Hog1 promotes Rga1 activation by blocking its Cdk1-mediated phosphorylation, thereby allowing its phosphoprotein phosphatase 2A (PP2A)-mediated dephosphorylation. These findings shed light on why Hog1 activity is required to prevent crosstalk from the HOG pathway to the mating pheromone response pathway.Eukaryotes utilize distinct mitogen/messenger-activated protein kinase (MAPK) pathways to evoke appropriate responses when confronted with different stimuli. In yeast, hyperosmotic stress activates MAPK Hog1, whereas mating pheromones activate MAPK Fus3 (and MAPK Kss1). Because these pathways share several upstream components, including the small guanosine-5'-triphosphate phosphohydrolase (GTPase) cell-division-cycle-42 (Cdc42), mechanisms must exist to prevent inadvertent cross-pathway activation. Hog1 activity is required to prevent crosstalk to Fus3 and Kss1. To identify other factors required to maintain signaling fidelity during hypertonic stress, we devised an unbiased genetic selection for mutants unable to prevent such crosstalk even when active Hog1 is present. We repeatedly isolated truncated alleles of  Multiple MAPK pathways enable a eukaryotic cell to respond appropriately when exposed to diverse stimuli. These signaling cascades often share multiple components ,2, raisiSaccharomyces cerevisiae) are subjected to hypertonic conditions, the MAPK Hog1 is activated via either of two routes that converge on an upstream MAPK kinase (MAPKK) Pbs2 . Samples were boiled for 5 min and then clarified by centrifugation for 5 min at max speed in a microfuge. HA-Rga1 electrophoretic mobility shifts were assessed using 10% acrylamide gels with an acrylamide-to-methylene-bis-acrylamide ratio of 75:1. All other protein samples were resolved using standard 10% gels (acrylamide-to-methylene-bis-acrylamide ratio of 29:1). Proteins were transferred to nitrocellulose, blocked with OdysseyTM blocking buffer  for 1 h, and probed with the desired antibodies overnight at 4 °C. Immunocomplexes on the blots were visualized by incubation for 2 h with an appropriate infrared dye-coupled secondary antibody diluted 1:10,000 in 1:1 TBS:OdysseyTM block containing 0.1% Tween and 0.02% SDS and, after thorough washing, imaged using an OdysseyTM infrared scanner (Li-cor Biosciences). Antibodies used were: mouse monoclonal α-HA.11  diluted 1:1000 in 1:1 TBS:OdysseyTM block + 0.1% Tween-20; α-Pgk1  containing EDTA-free Complete Protease Inhibitors , incubated on ice for 30 min and lysed by passage through a French pressure cell at 20,000 PSI twice. Lysates were clarified by centrifugation at 12,000× g for 10 min. To the resulting supernatant fraction, 0.5 mL of a 50:50 slurry of glutathione-Sepharose 4B beads  (prewashed in 5 mL cold 1X PBS) was added and incubated for 1 h at 4 °C. Beads were washed with 3 × 5 mL Protease 3C buffer , followed by the addition of 1 mL Protease 3C buffer and 40 units of PreScission Protease . Proteins were eluted by 3C cleavage at 4 °C for 18 h, and then dialyzed against dialysis buffer  overnight at 4 °C.Hog1-6XHIS, catalytically-inactive Hog1-KD-6XHIS, Hog1-as-6XHIS, and GST-Pbs2-EE proteins were purified and in vitro protein kinase assays with , suspended in 40 µL phosphatase buffer and split in half. Calf intestinal alkaline phosphatase   was added to one of the aliquots, and both samples incubated at 30 °C for 1 h. After incubation, 4X SDS-PAGE loading buffer was added  and samples boiled 5 min prior to SDS-PAGE and immuno-blotting.Cells pellets (from 40 mL culture) were resuspended in 200 µL cold immunoprecipitation (IP) lysis buffer .Hog1 activity is necessary to prevent crosstalk ,57; therproteome ,99,100. and T571 , in agreand T571 . In our and T571 . The Hogand T571 ; reassurrga1-505 allele. For that reason, using site-directed mutagenesis, we constructed a Rga1(T470A T532A T541A T571A) mutant and integrated the DNA sequence encoding it into the genome in place of the normal RGA1 locus. We found that the resulting strain did not display any elevated crosstalk to the mating pathway (data not shown). We conclude that, although Hog1 is capable of modifying Rga1, direct phosphorylation of Rga1 is not how Hog1 function promotes Rga1-mediated squelching of crosstalk.If the mechanism by which Hog1 action promotes the inhibition of crosstalk is by direct phosphorylation and stimulation of Rga1, then those modifications would presumably be necessary to promote the ability of Rga1 to deactivate GTP-Cdc42. If so, we anticipated that a Rga1 mutant lacking its most prominent Hog1 sites would be unable to prevent crosstalk, like the 561P-) is highly conserved in Rga2 (S. cerevisiae phosphoproteome [541P-), i.e., juxtaposed to a basic residue, matches that of three other Cdk1/Cdc28 phosphorylation sites previously mapped in both Rga1  [733P- and -S772PKR-) [cdc28-1) [Because of its large size (113 kDa), it was technically challenging to monitor the phosphorylation status of epitope-tagged Rga1 in vivo by electrophoretic mobility shift; nonetheless, we were able to devise conditions to do so. During exponential growth in normal medium, HA-Rga1 runs primarily as two isoforms of approximately equal intensity A. Within in Rga2  and has proteome . MoreoveHS331P-)  and in R772PKR-) ,103. Indcdc28-1)  to non-pCurrent evidence indicates that the Cdk1-dependent phosphorylation of both Rga1  and Rga2sic1∆ and swe1∆ single mutants and even in a sic1∆ swe1∆ double mutant  and carrying a hypomorphic allele in a third PP2A catalytic subunit (pph22-12) [We found, however, that hyperosmotic shock still induced the HA-Rga1 mobility shift in e mutant D. Hence,llection  that affph22-12) , hypertoIn summary, we conclude from our findings that, upon hyperosmotic shock, inhibitory Cdk1-mediated phosphorylation of HA-Rga1 is alleviated and thereby its GAP function activated because activated Hog1 prevents Cdk1-mediated phosphorylation of Rga1, allowing for its efficient PP2A-mediated dephosphorylation. Activated Rga1, in turn, down-regulates the available pool of GTP-Cdc42, preventing crosstalk G.SHO1 dependence of mating pathway activation by hypertonic stress. Mechanisms for negative feedback within the HOG pathway are invoked to explain how Hog1 action prevents crosstalk, including phosphorylation and down-regulation of Sho1 [Here we used a genetic selection to identify Cdc42-GAP Rga1 as a key gene product required to squelch crosstalk between the Hog1 pathway and the Fus3 (and Kss1) MAPK pathways. A chief feature was the ability to screen candidate mutants for the  of Sho1 ,109 and  of Sho1 ,111. By  of Sho1 .By acting on Cdc42-GTP generated locally by the HOG pathway, and because the ability of Cdc42 to diffuse away may be hindered due to association with other proteins in the complex and to its anchoring in the membrane by its geranylgeranylation , Rga1 isrga1-505 allele  increases crosstalk more than it elevates signal flux in the HOG pathway. In ssk1∆ cells, 20.3% of the total Hog1 was activated after a 20 min treatment with 1 M sorbitol, whereas 62.6% was activated in ssk1∆ rga1-505 cells under the same condition , whereas 18.9% was activated in ssk1∆ rga1-505 cells , there was especially potent expression of the mating pathway reporter when the Hog1-as enzyme was inhibited in Rga1-deficient cells. The mating pathway reporter activation that occurred under these conditions was entirely Sho1-dependent. These findings indicate that, normally, even the basal signaling emanating from the Sho1 branch of the HOG pathway is kept in check by the joint actions of Rga1 and Hog1. Thus, when both are removed, crosstalk can occur unimpeded.ssk1∆ cells carrying a rga1∆ and especially a rga1-505 mutation, response to modest hypertonic shock was almost fully restored, indicating that Rga1 sets the threshold for response by the Sho1 branch. Moreover, as observed by others [Aside from its role in preventing crosstalk, Rga1 seems an important regulator of the Sho1 branch of the HOG pathway, a conclusion also reached by others using a candidate gene approach . As we sy others  and confy others .In keeping with its multiple roles in vivo, we found phospho-regulation of Rga1 to be complex. Although Hog1 may phosphorylate Rga1 directly, we found that the primary effect of hyperosmotic shock was to cause a Hog1-dependent dephosphorylation of the Cdk1-mediated phosphorylation of Rga1. Moreover, although the transient Hog1-mediated inhibition of Cdk1 described by others may contribute, we found that the resulting slowed rate of Cdk1-mediated phosphorylation simply allowed for more efficient PP2A-mediated dephosphorylation of Rga1. There is no evidence of which we are aware that Hog1 actively stimulates PP2A function; on the other hand, activation of PP2A in mammalian cells by p38, the functional ortholog of Hog1 , has beeHow two MAPK pathways that respond to different stimuli, yet share multiple components in common, can generate distinct outputs, has been unclear until now. In this study, we demonstrate that a Cdc42-specific GAP, Rga1, negatively regulates the Sho1 branch of the HOG pathway and also deactivates GTP-bound Cdc42 down to a level insufficient to evoke another Cdc42-dependent MAPK pathway, the mating pheromone response pathway. Thus, this regulatory circuitry blocks inappropriate pathway crosstalk by a kinetic proof-reading mechanism.


Analysis of diffraction data from three proteins indicates that the ice formed in internal crystal solvent is stacking-disordered. Application of a revised metric and algorithm for detecting ice from protein structure-factor data indicates that roughly 3.9% of PDB entries exhibit ice that is primarily hexagonal and 11.7% exhibit stacking-disordered ice. et al. (2017Acta Cryst. D73, 729–727], a revised metric is defined for detecting ice from deposited protein structure-factor data, and this metric is validated using full-frame diffraction data from the Integrated Resource for Reproducibility in Macromolecular Crystallography. Using this revised metric and improved algorithms, an analysis of structure-factor data from a random sample of 89 827 PDB entries collected at cryogenic temperatures indicates that roughly 16% show evidence of ice contamination, and that this fraction increases with increasing solvent content and maximum solvent-cavity size. By examining the ice diffraction-peak positions at which structure-factor perturbations are observed, it is found that roughly 25% of crystals exhibit ice with primarily hexagonal character, indicating that inadequate cooling rates and/or cryoprotectant concentrations were used, while the remaining 75% show ice with a stacking-disordered or cubic character.Diffraction data acquired from cryocooled protein crystals often include diffraction from ice. Analysis of ice diffraction from crystals of three proteins shows that the ice formed within solvent cavities during rapid cooling is comprised of a stacking-disordered mixture of hexagonal and cubic planes, with the cubic plane fraction increasing with increasing cryoprotectant concentration and increasing cooling rate. Building on the work of Thorn and coworkers [Thorn  al. 2017, Acta Cr When such a peak overlaps with a protein diffraction peak, the measured intensity will be larger than those of symmetry-related reflections and/or will be larger than that predicted from Wilson statistics , has been developed and implemented within the et al., 2016et al., 2019Here, we examine the nature and effects of ice on diffraction data in protein cryocrystallography. We first examine ice diffraction in diffraction frames that we have collected from crystals of three proteins and in reference diffraction-frame data obtained from the Integrated Resource for Reproducibility in Macromolecular Crystallography . Our approach obtains more accurate ice detection with far fewer false positives and false negatives and is based on a p-value testing the null hypothesis that the structure factors are not biased by ice diffraction. Using our revised ice-detection metric and improved algorithms, we find that roughly 16% of PDB entries show structure-factor perturbations from ice contamination, consistent with the results of Thorn and coworkers. Of these, roughly 25% show evidence of ice contamination at the positions of all-hexagonal ice peaks, and for the remaining 75% the observed peaks are consistent with stacking-disordered ice with a substantial cubic fraction.We then extend the methods developed by Thorn  al. 2017 for dete2.2.1.et al.  glycerol for at least 5 min. Crystals were then transferred to a separate drop of NVH oil (Cargille) and manipulated until all external solvent was removed from their surface, as indicated by a near-disappearance of the crystal due to the close match between its refractive index and that of the oil  using a PILATUS 6M detector. A cold nitrogen-gas stream (Oxford Cryosystems Cryostream 700) with a flow rate of 5 l minSupplementary Fig. S1 shows an image of the sample in the beam path.X-ray diffraction data from cryocooled glycerol/water solutions were collected using the same CHESS station and experimental protocols. Samples were prepared by injecting ∼10 nl of solution into a long thin-walled polyester tube of 250 µm in diameter and ∼2 cm in length, resulting in a ∼200 µm long plug of liquid. The tube was then affixed to a goniometer base (MiTeGen GB-B1A) and centred in the X-ray beam. 2.3.Supplementary Section S2.Discrete ice diffraction peaks, associated with diffraction from a single ice crystal, were identified by searching for pixels that were located near expected ice-ring positions and were not associated with protein diffraction, having values approximately five times larger than the local background intensity. The detailed algorithm used is described in 2.4.h), cubic (Ic), a mixture of these crystal forms (Ih + Ic) or stacking-disordered (Isd), in which cubic and hexagonal planes are randomly stacked, as shown in Supplementary Fig. S2   from the sensitivity , which measures the ability to correctly identify entries with ice biasing, and their specificity , which measures the ability to correctly identify entries that are ice-free  containing pools of solvent that are larger than the solvent cavities in ordered protein crystal regions. This fraction has increased by roughly 60% over the last 20 years. Diffraction from this hexagonal ice tends to be anisotropic and inhomogeneous, unlike diffraction from the stacking-disordered ice that forms in the solvent cavities of reasonably well ordered crystals. As a result, hexagonal ice diffraction tends to be much more difficult for advanced background-subtraction methods to account for and so has a larger impact on the integrated structure factors.For the broader PDB, both et al., 2004The increasing prevalence of hexagonal ice, even as beamline cryocooling hardware has evolved to largely eliminate frosting, suggests that the cryocooling protocols for an increasing fraction of structural targets have been inadequate. This could reflect a greater focus on challenging targets: crystals with large solvent contents, large solvent cavities, fragile lattices, inconvenient  growth habits and/or for which suitable cryoprotectant conditions may be difficult to identify. Time-consuming cryoprotection protocols may have been relaxed to increase throughput, and a larger fraction of crystallographic data are now collected by those for whom crystallography is not a primary focus. The shift to remote data collection may also be a factor. While a dry shipper does an excellent job at keeping crystals cold in transit to a synchrotron facility  and whether gas or liquid cryogens are used  Cryoprotectant concentration. In aqueous solutions, the minimum cooling rates required to obtain a sample with no detectable ice increase exponentially with decreasing cryoprotectant concentration  The amount of solvent surrounding the crystal. Ice formation in solvent internal to the crystals is strongly suppressed by nanoconfinement  Crystal solvent content and solvent-cavity size. Ice formation in internal solvent is strongly suppressed by nanoconfinement, and solvent within the first two hydration layers adjacent to the protein molecules generally does not crystallize. Ice is thus most likely to form within crystals with large solvent cavities and large fractions of bulk-like internal solvent  Crystal perfection. Growth defects such as dislocations, inclusions and vacancies, more general lattice-scale disorder caused by imperfect molecular packing, as well as defects/disorder created by osmotic shock during cryoprotectant soaks, inadvertent crystal dehydration and mechanical damage during handling, can all produce solvent pockets within the crystal that are much larger than the solvent cavities in ordered portions of the crystal that are identified by crystallography. Solvent in these relatively less confined regions has a higher freezing point  We conclude by summarizing the factors that affect ice formation in cryocrystallography (Garman, 19995.supporting information for this article: Fortes (2018The following reference is cited in the rtes 2018.10.1107/S2059798321001170/tz5104sup1.pdfSupporting Information and Supplementary Figures and Tables. DOI: Click here for additional data file.10.1107/S2059798321001170/tz5104sup2.xlsxExcel workbook comparing the performance of our ice-detection methods with those of AUSPEX for ground-truth and test data sets. DOI: Click here for additional data file.10.1107/S2059798321001170/tz5104sup3.zipPlots of deposited structure-factor intensities, IFS, DS and N versus resolution for the 200 PDB entries also used by Thorn and coworkers to benchmark their algorithm. DOI: Click here for additional data file.10.1107/S2059798321001170/tz5104sup4.zipScripts for P_Ice. The example generates some results from this paper. DOI:


Ancient glaciated landscapes reveal interactions among ice dynamics, bed composition, and preglacial river networks. Subglacial landscapes, revealed in regions of recent ice-sheet retreat, provide a window into ice-sheet dynamics and interactions with evolving subglacial topography. Here, we document landscape evolution beneath the southern Laurentide Ice Sheet of North America since the end of the Pliocene, 2.6 million years (Ma) ago, by reconstructing the isostatically adjusted preglacial surface and modern bedrock topography at 250 m horizontal resolution. We use flow routing to reconstruct drainage networks and river longitudinal profiles, revealing the pattern and extent of their glacially forced reorganization. The overall mean Quaternary (2.6 Ma ago to present) erosion rate is 27 m/Ma, rising within ice-streaming corridors to 35 m/Ma  and falling to 22 m/Ma in non–ice-streaming regions. Our results suggest that subglacial erosion was sufficient to lower the southern Laurentide Ice Sheet into warmer environments, thereby enhancing ablation and reducing ice-sheet extent over time. During the Quaternary, the Laurentide Ice Sheet (LIS) repeatedly advanced across the North American mid-continent, modifying the landscape and depositing low-relief and agriculturally productive sediments across the north-central United States and southern Canada. Buried beneath these deposits lies a hidden topography of river gorges, drainage divides, and glacially scoured bedrock that records the past 2.6 million years (Ma) of landscape evolution . This is almost double the average continental erosion rate (16 m/Ma) during the Phanerozoic , and the highest rates of erosion are dominantly or only seen here , bracketed by mean deposit thicknesses of 38 m in ice-streaming regions and 40 m outside of them. Deep scours, where erosion:deposition ratios can exceed 10:1 , may forMost reconstructed rivers in our end-Pliocene DEM bear concave-upward longitudinal (long) profiles, typical of graded rivers  (32). FuIce advance rerouted northward- and eastward-flowing rivers by damming them, forming lakes that overtopped and incised drainage divides (fig. S9) with the aid of abundant glacial meltwater  state and provincial maps. These disparate datasets of buried bedrock surfaces (figs. S1 and S2) are available in either digital or analog map formats from most states and provinces within the Laurentide region. We applied three approaches to convert multiple bedrock-elevation data sources to elevation contours in digital vector formats for use in a surface interpolation algorithm and summarize these methods in fig. S1. (i) In the “direct-use” approach, we directly ingested and integrated existing digital contours and bedrock DEMs that we converted to digital contours. (ii) Using a “refinement” approach, we integrated local data with edge-matching datasets  to align valley and ridge features and/or augment base datasets with additional information from published work. An example of this refinement step was to incorporate Great Lakes bedrock contours from academic publications in place of the default lake bathymetry. (iii) Last, the absence of bedrock-elevation data in some areas required us to interpolate existing point bedrock elevations to produce bedrock-surface topographic contours at a resolution consistent with our larger-scale dataset. We obtained the depth of the bedrock interface from drilling logs and prepared data for interpolation  were used to obtain an average root mean square error of 12.5 m .We generated the complete bedrock surface by assimilating different elevation data types, such as points and polylines (contours), from the sources described above. To do this, we used the ANUDEM gridding algorithm  were analyzed to define geomorphic features such as river terraces and truncated spurs (eroded ridgelines) and estimate local channel segment elevations. We interpolated channel elevations along with interfluve elevation constraints extracted from the bedrock surface DEM based on previous literature  end-Pliocene DEM elevations along each of these five rivers; extracting elevations from the unmodified DEM produces some of the irregularities visible in We extracted river long profiles from the paleo-Saskatchewan, Missouri, St. Lawrence, Platte, and Wabash Rivers . We floo


TM screening platform, and we identified casein kinase 2 (CK2) as a novel target. KD025 showed comparable binding affinity to CK2α (Kd = 128 nM). By contrast, CK2 inhibitor CX-4945 and ROCK inhibitor fasudil did not show such cross-reactivity. In addition, KD025 effectively inhibited CK2 at a nanomolar concentration (IC50 = 50 nM). We examined if the inhibitory effect of KD025 on adipocyte differentiation is through the inhibition of CK2. Both CX-4945 and KD025 suppressed the generation of lipid droplets and the expression of proadipogenic genes Pparg and Cebpa in 3T3-L1 cells during adipocyte differentiation. Fasudil exerted no significant effect on the quantity of lipid droplets, but another ROCK inhibitor Y-27632 increased the expression of Pparg and Cebpa. Both CX-4945 and KD025 acted specifically in the middle stage (days 1–3) but were ineffective when treated at days 0–1 or the late stages, indicating that CX-4945 and KD025 may regulate the same target, CK2. The mRNA and protein levels of CK2α and CK2β generally decreased in 3T3-L1 cells at day 2 but recovered thereafter. Other well-known CK2 inhibitors DMAT and quinalizarin inhibited effectively the differentiation of 3T3-L1 cells. Taken together, the results of this study confirmed that KD025 inhibits ROCK2 and CK2, and that the inhibitory effect on adipocyte differentiation is through the inhibition of CK2.KD025, a ROCK2 isoform-specific inhibitor, has an anti-adipogenic activity which is not mediated by ROCK2 inhibition. To identify the target, we searched binding targets of KD025 by using the KINOMEscan Rho-associated coiled-coil-containing protein kinases (ROCKs/Rho-kinases) are serine/threonine kinases introduced as downstream binding partners of RhoGTP protein ,2. To daIn cell culture model system, Rho/ROCK activity negatively regulates adipocyte differentiation. Unspread and round cell shape ,11 and i50 = 105 nM (24 µM for ROCK1)  and fasudil were purchased from Medchem Express , Y-27632 from Selleck Chemicals . CX-4945 and DMAT were provided by Medchem Express . Quinalizarin  was purchased from Sigma-Aldrich .TM screening platform , which is an active site-directed competition binding assay to measure quantitatively the interactions between test compounds and kinases. This platform contains 468 recombinant human kinases (more than 80% coverage) . Then, 20 μg lysate was separated by SDS-PAGE. The separated proteins were transferred from the gel to a nitrocellulose membrane. The membranes were incubated with primary antibodies ; CK2α was from Cell Signaling. The probed membranes were incubated with the secondary antibodies. The bands were detected using Amersham ECL Western Blotting Detection Reagent  and visualized and quantified using the ChemiDoc imaging system .Total protein was extracted from cells using lysis buffer [20 mM tris pH 7.5, 5 mM EDTA, 10 mM Nap < 0.05; ** p < 0.01; *** p < 0.005 vs. vehicle.Data for ORO staining, qRT-PCR, and immunoblot were the representatives from three independent experiments. Data are expressed as means ± S.D. based on duplicate or triplicate. *


CaMKII and CaMKIV are calcium/calmodulin-dependent kinases playing a rudimentary role in many regulatory processes in the organism. These kinases attract increasing interest due to their involvement primarily in memory and plasticity and various cellular functions. Although CaMKII and CaMKIV are mostly recognized as the important cogs in a memory machine, little is known about their effect on mood and role in neuropsychiatric diseases etiology. Here, we aimed to review the structure and functions of CaMKII and CaMKIV, as well as how these kinases modulate the animals’ behavior to promote antidepressant-like, anxiolytic-like, and procognitive effects. The review will help in the understanding of the roles of the above kinases in the selected neurodegenerative and neuropsychiatric disorders, and this knowledge can be used in future drug design. However, to fulfill its role, the Ca2+ signal has to be suitably processed. The process is performed by sensor proteins, such as calmodulin (CaM), which can specifically complex Ca2+. CaM is a major Ca2+-binding protein in the central nervous system. The molecule has four similar domains, each containing one Ca2+-binding site. After binding to CaM, Ca2+ alters its conformation, and the resulting CaM–Ca2+ complex can interact with target proteins and modulate their activity. The major CaM target proteins are Ca2+/CaM-dependent kinases (CaMKs), which contribute to many neuronal regulatory pathways, from synaptic plasticity to gene transcription , contains ATP- and substrate-binding site. The reaction of substrate phosphorylation runs within this domain .2+/CaM-binding site) in inactivated state interacts with the catalytic domain, limiting access to the ATP-binding site, and also prevents Ca2+/CaM-independent phosphorylation. Although it was crystallized in helical conformation, the autoinhibitory domain in the basal state forms α helix only in the N-terminal part. When activated by Ca2+/CaM, it changes conformation enabling the activity of the kinase. This domain contains Thr268, the primary site of autophosphorylation [Regulatory or autoinhibitory domain  forms the hub complex of holoenzyme (described in the following section). It acts as a flexible linker region that facilitates the formation of a variety of conformational states, which usually incompletely match the average reconstruction model (including asymmetric forms) .From the day of the first discovery of CaMKII, efforts were made to establish the three-dimensional structure of its holoenzyme. Various arrangements were suggested, e.g., two parallel hexametric rings ,53,56,57Typical neuronal holoenzyme is built of α and β subunits, 12 in total, which create homo- or heteropolymeric molecules in the various quantitative ratio (depending on investigated tissue). For instance, the majority of forebrain enzyme molecules possess 10 petals of α subunits only, and the cerebellum enzyme mostly consists of eight petals, β subunits only ,46,58. The inactive state of the kinase is guarded by the autoinhibitory domain that is positioned to sterically occlude the substrate-binding site and to disrupt ATP binding. Two neighboring autoinhibited catalytic domains of the holoenzyme create symmetric dimer that is held together by a coiled coil formed by their regulatory domains ,58. Holo2+/CaM complex attaches to the binding site within the regulatory domain, which changes the conformation of its C-terminus into helical and pushes the N-terminal part into disordered conformation. It causes separation of previously dimeric catalytic domains and starts the intersubunit reaction. Subunit activated by Ca2+/CaM acts like a kinase, and adjacent subunit is its substrate. Exposed Thr286 undergoes phosphorylation, and anionic phosphate moiety blocks reformation of the coiled coil and maintains dissociation of a catalytic domain from its regulatory domain. This extended conformational change disables inhibition of ATP binding, enables phosphorylation of various substrates, consolidates affinity of CaM to autophosphorylated kinase , and enhances the binding of active kinase to synaptic sites [2+ drop and Ca2+/CaM dissociates. Taken together, activation through autophosphorylation of Thr286 is Ca2+/CaM-dependent, but it results in partially Ca2+/CaM-independent kinase activity by reducing the effectiveness of inhibitory domains [Activation starts when the Caic sites ,59. Afte domains ,54.2+-independently. It blocks CaM rebinding and favors dissociation of CaMKII from protein anchors in the synaptic site [The autoregulation cycle ends up in secondary autophosphorylation of Thr305/Thr306  occurring Catic site ,46.2+ oscillations into distinct levels of enzyme activity in vitro and may play a similar role in vivo [Autophosphorylation is supposed to promote high-affinity CaM binding  by enabling favorable interactions between Glu120 and Met124 of CaM and residues 293-298 of the kinase. When CaMKII is unphosphorylated, Phe293 and Asn294 have strong connections to Phe98 and Ile205 from the catalytic site. Thus, Phe293 and Asn294 either stabilize the autoinhibited form of the enzyme or, when Thr286 is phosphorylated, consolidates the CaM-bound form. CaM trapping prolongs kinase activity and prevents immediate autophosphorylation of Thr305/Thr306. It is also likely the ground for the ability of CaMKII to decode the frequency of Ca in vivo .2+. In general, if the concentration of Ca2+ is high, Thr286 autophosphorylation spreads rapidly through subunits of the holoenzyme, leading to the onset of Ca2+/CaM-independent activity. When Ca2+ level falls, Thr286 is usually dephosphorylated before activation can proceed [2+ oscillations. In this respect, the arrangement of the subunits in a holoenzyme is crucial. Appropriate pulsatile activation of CaMKII may extend its activity way beyond the period of Ca2+ elevation. This feature may play an important role in frequency-dependent changes in synaptic plasticity [As proved in previous sections, the activity of CaMKII is closely linked to calmodulin and Ca proceed . Howeverasticity ,61. 2+/CaM-dependent activity [2+/CaM-independent activity [The autophosphorylation occurs at several other sites of CaMKII, and one of these is Thr253. This exact process is relative to Thr286 autophosphorylation, favored by low concentration of ATP and increases in response to depolarization. It enhances holoenzyme binding to proteins in subcellular fractions enriched in PSDs (post-synaptic densities) but does not affect autonomous activity nor Caactivity . SeveralCaMKII targeting multiple anchoring proteins can occur thanks to several distinct interactions. For example, the association domain that has isoform-specific inserts may bind the β-isoform to actin filaments. The Thr286 docking site, exposed by the activation of the kinase, is used to anchor the enzyme to the NMDA receptor subunit NR2B. This way, interactions between domains of CaMKII and multiple anchoring proteins may lead to clustering of holoenzymes to a particular site, such as the synapse . CaMKII is expressed throughout the body, phosphorylates a wide range of substrates, and regulates numerous cellular functions. CaMKII regulates cardiac functioning , 2+ currents, CaMKII regulates the neurotransmitter release [CaMKII controls various neurotransmitters systems; however, the presynaptic CaMKII may act as either enhancer or inhibitor of neurotransmitter release depending on properties of specific synapses ,80,81,82 release . During neuronal development, the proper CaMKIIβ functioning is required for the correct neuronal migration . This isThe CaMKIV was first described as ‘CaM-kinase GR’ because of its abundance in the cerebellum’s granular cells . There aThe gene of 42 kbp encoding the enzyme consists of 12 exons and 11 introns and is considered as encoding both α and β forms of kinase as well as calspermin—a testis-specific calmodulin-binding protein. These are all produced by alternative sites of transcription initiation. CaMKIVβ comes from the longest transcript. The promoter of CaMKIVα is located within the first intron, and calspermin has its promoter and first exon located in intron 10 . When thThe subunit is comprised of the protein kinase domain (residues 42–296), autoinhibitory domain 297–336), overlapping PP2A-binding domain (302–319), and CaM-binding domain (318–337). The most significant site of phosphorylation is Thr196 (numbering refers to rat isoform) [–336, oveThe N-terminal part of the human CaMKIV is highly conserved compared with rat and mouse parts. In contrast, the C-terminal part is very less conserved (≈37% sequence identity) with rat and mouse CaMKIV. These kinases possess a parallel sequence motif, X-Arg-X-X-Ser/Thr, with X meaning a hydrophobic residue. Altogether, it creates an essential site for phosphorylation . The first 291 amino acids of CaMKIV have been found to be 58% homologous to the first 314 amino acids of CaMKIIα (if conservative amino acid substitutions are considered). The entire amino acid sequences are 42% homologous .Within the large lobe of the kinase domain, an activation loop with Thr196 is located. This site needs to be phosphorylated for proper folding of the ATP-binding pocket and optimal activity of kinase .2+/CaM binding to the regulatory segment enables Thr196 phosphorylation by CaMKK. It results in conformational changes that abolish the interaction between the catalytic core and autoinhibitory domain and generation of highly active form of CaMKIV, partially Ca2+/CaM-independent. The active site becomes fully exposed for substrate binding and its phosphorylation. The third level of activation concerns autophosphorylation of serine residues at the N-terminal part of CaMKIV, Ser11, and Ser12. In the resting state autoinhibitory sequence interacts with the catalytic domain. This inhibition is not competitive with ATP but with the peptide substrate ,95. Ca2+2+ level decreases significantly, it is followed by the dissociation of Ca2+/CaM and PP2A binding, which causes dephosphorylation of CaMKIV and its deactivation [When the Cativation ,99,100.CaMKIV undergoes several posttranslational modifications, one of which is GlcNacylation. The addition of O-GlcNac to Ser/Thr residues participates in regulating the activation and function of CaMKIV . One of the factors responsible for targeting CaMKIV may be polyglutamate sequences present within the C-terminal part of the kinase. They are supposed to target the enzyme to the nuclear chromatin of specific neurons .CAMKIV also controls various biological activities starting with the regulation of the activity of the numerous transcription factors such as cAMP-response element binding protein (CREB), myocyte enhancer factor 2D, JUN, ATF-1, SRF, and retinoic acid-related orphan receptor-alpha ,66,67,68Since both CaMKII and CaMKIV are widely distributed across the nervous system, they participate in many crucial neuronal processes. Their abnormal functioning induces negative changes and consequently may lead to the diseases’ development. Here, we discuss the role of CaMKII and CaMKIV in depression and anxiety , whose pDepression is a devastating mental disorder due to abnormal changes in different brain areas such as the hippocampus or lateral habenula . The expCaMKII is the kinase involved in the pathophysiology as well as in the antidepressant activity of drugs. CaMKIIα knockout mice did not show any changes in behavior during the forced swim test, a test used to evaluate antidepressant-like effects in rodents . MoreoveVarious animal models of depression induce changes CaMKIIβ expression in several brain structures. CaMKIIβ level was increased in congenitally learned helpless rats, after the acute learned helplessness, chronic mild stress, or lipopolysaccharide injections ,119. MorThe CaMKII is a kinase significantly implicated in the mechanism of antidepressant drugs’ action. The common finding is that long-term treatment with antidepressants is connected with adaptive and plastic changes in molecular and cellular neuronal signaling . In the Some researchers indicated that chronic administration of fluoxetine may cause the epigenetic control of CaMKIIα promoter in nucleus accumbens, influencing the transcription factor ΔFosB binding in animals .Moreover, CaMKII is essential for the antidepressant-like action of ketamine. CaMKII inhibitor prevented the ketamine-induced decrease in immobility in the forced swim test in mice . SimilarOn the other hand, the phosphorylation of CaMKII in the hippocampal region CA1 of olfactory bulbectomized mice was increased after repeated administration of sunifiram, a novel pyrrolidone nootropic drug, but the treatment did not ameliorate depressive-like behavior in the tail suspension test in animals . InteresThere are also studies with Guanxin Danshen formula, which is a natural botanical drug composition with potential neuroprotective and antidepressant-like activity. Xie and colleagues confirmed the antidepressant-like effect of Guanxin Danshen formula in rats exposed to the unpredictable chronic mild stress . They obAnimal studies also suggested that antidepressant-like effects of deep brain stimulation depend on CaMKII phosphorylation in lateral habenula as well as its downregulation in the infralimbic cortex . MoreoveTaken together, we can generalize that different isoforms of CaMKII play different roles in depressive-like phenotype and antidepressant response. The increase in CaMKIIβ and decrease in CaMKIIα are linked with depressive-like phenotype in animals . MoreoveCaMKIV is one of the key regulators of different intracellular processes such as neuroprotection and neuroplasticity . There aCaMKIV expression is altered in various animals’ models of depression. Patki and colleagues showed that social defeat stress in rats reduced the level of CaMKIV in the hippocampus, leading to a depressive-like state in animals assessed by the sucrose preference test . SimilarA number of drugs, compounds, or therapies elicit their antidepressant-like activity probably via CaMKIV pathway’s modulation. In CaMKIV knockout mice, chronic nicotine treatment decreased depressive-like behavior via alfa7-nAChRs (nicotinic acetylcholine receptors) and subsequent activation of this signaling pathway . The revStudies of hyperforin, which is an active constituent of St. John’s wort extract with documented antidepressant activity, indicated that the CaMKIV is one of the signaling pathways that mediates the improvement of synaptic plasticity via TRPC6  channels activation) and probably explains the pharmacological effects of hyperforin .Presented data confirmed the relation of CaMKIV with the pathophysiology of depression and allowed us to consider CaMKIV as a potential target for novel antidepressant therapies. The CaMKIV deletion is linked to a depressive-like state in animals, and many studies reported the decreased kinase phosphorylation in animal models of depression. Thus, we may conclude that CaMKIV activation is preferable for antidepressant-like activity.Anxiety is a mental disorder leading to excessive nervousness, fear, and apprehension, and its prevalence increases worldwide . Given tDeletion of genes coding CaMKII isoforms led to specific changes in phenotype, and anxiety-like responses in particular. CaMKIIβ knockout mice exhibited decreased anxiety-related behavior assessed during the elevated plus maze test and the open field test . In bothCaMKII role in the regulation of fear-related phenotype is more complex and more profound than we have thought. Exposure of rats to predator odor induced the defensive burying response and the activation of CaMKII-positive subpopulations of the amygdala . MoreoveMoreover, the phosphorylation of CaMKII can be regulated via various receptors or enzymes. Mice without soluble epoxide hydroxylase exhibit anxiety-like behavior as a result of the hyperphosphorylation of CaMKII and glycogen synthase kinase 3 α/β (GSK3α/β) . MoreoveInterestingly, CaMKII plays a crucial role in the 5-HT1A-mediated pathways determining the anxiety-like response during the developmental period . The 5-HThe CaMKII inhibitors are an attractive therapeutic option. TatCN21 peptide (CaMKII inhibitor) reversed the anxiety-like state in the elevated plus maze test and open field test in rats with global cerebral ischemia . Thus, tTo sum up, CaMKII participates in mediating the anxiolytic-like response in animals. The depletion of both α and β isoforms causes an anxiolytic-like effect, whereas their overexpression leads to an anxiety-like state . MoreoveCaMKIV is known for being involved in behavioral alterations, mainly by phosphorylating a significant transcription factor—CREB and CRE modulator (CREM). CaMKIV knockout mice showed a decrease in anxiety-like response in common behavioral paradigms such as elevated plus maze test, dark–light emergence test, light/dark box, and acoustic startle and prepulse inhibition . This suThe alternation in CaMKIV level is also seen in animals subjected to stress . Social On the other hand, the role of CaMKIV in anxiety seems to be region-specific. Mice with dominant-negative CaMKIV variant in nucleus accumbens elicited anxiogenic behavior in the light/dark test . That suInterestingly, the CaMKIV influences anxiety-like behavior in drug- or substance-dependent animals. The research on nicotine dependence and withdrawal in CaMKIV knockout mice was conducted using a conditioned place preference test . ResultsThe main conclusion from the above studies is that the depletion of CaMKIV is responsible for the anxiolytic-like effect in animals . HoweverBoth CaMKII and CaMKIV are the important nexuses of the memory and learning processes, and their proper functioning is required to remember longer the events or things. Moreover, memory deficits occur more often due to increased life expectancy and accompany mood disorders . Here, wCaMKII phosphorylation is critical for long-term potentiation (LTP) induction in the CA1 region of the hippocampus ,193,194.The level of phosphorylated CaMKII depends on age and memory training. Moreover, the kinase amount varies across different brain structures . ContextIn CaMKIIα knockout and CaMKIIα-T286 mutant mice (with blocked autophosphorylation at threonine-286), scientists observed impaired spatial memory with a concomitant decrease in LTP formation ,168,169.CaMKIIα depletion in mice is also related to impaired contextual fear memory, manifesting in reduced freezing responses in the fear conditioning task ,167. HowNotably, the CaMKII expression’s location also plays a vital role in memory processes ,203. TheThe interesting issue raised concerns about the role of CaMKII in the learning process and separately in memory. In three amygdala-dependent tasks , lack of CaMKIIα autophosphorylation led to learning retardation. However, extended training normalized the behavioral outcomes of both wild-type and CaMKIIα-T286 mutant mice . The norThe CaMKII regulates the transcription of genes essential for memory consolidation via CREB (the upregulation of NGFI-B mRNA expression requires the autophosphorylation of CaMKIIα ) as wellThe decreased level of CaMKII in the frontal cortex and hippocampus occurs in various animal models of Alzheimer’s disease, such as amyloid precursor protein transgenic mice or vascular dementia gerbils ,210. MorHowever, CaMKII dysregulation may also modulate the toxicity in Alzheimer’s disease. Amyloid precursors protein and tau protein can be phosphorylated by hyperactive CaMKIIα, leading to Aβ and neurofibrillary tangles formation ,214,215.CaMKII is crucial for memory-improving properties of rivastigmine, sufiniram, or nefiracetam ,221,222.2+-exposed rats have decreased CaMKII activity, increased substrate affinity of the enzyme, disrupted LTP, and spatial learning deficits [CaMKII also plays a significant role in memory impairment caused by toxins, drugs, and heavy metals. Pbdeficits ,226. Mordeficits .Taken together, the CaMKII is a rudimentary kinase for learning and memory processes. In knockout animals, we observe disrupted emotional, recognition, spatial, and working memory . During CaMKIV plays a significant role in memory consolidation processes and well as long-term potentiation. CaMKIV knockout mice have disrupted synaptic plasticity on many levels . They haInterestingly, different results were reported regarding the function of CaMKIV in memory-assessing behavioral tasks. Genetic deletion of CaMKIV did not reflect on animals’ performance in spatial memory tests  ,166, whiNotably, CaMKIV controls not only spatial but also emotional memory. Both genetic depletion of CaMKIV and transgenic kinase inhibition lead to learning deficits in fear conditioning ,153,162.The long-term memory impairments are also seen in CaMKIV knockout mice performing eyeblink conditioning test  or durinOn the other hand, the overexpression of CaMKIV in the forebrain promotes social and fear long-term memory formation . Looking+-dependent Na+/Ca2+ exchanger) heterozygous mice, as well as attenuated hippocampal LTP with reduced CaMKII and CaMKIV activity [CaMKIV is an essential factor in the development of many cognitive deficits caused either by toxins, drugs, or age. The arsenic-induced decrease in CaMKIV expression subsequentially repressed cerebellar LTD formation, manifested by worse performance of mice in spatial memory task ,230. Theactivity . Moreoveactivity . Howeveractivity ,235. In activity . To conclude, CaMKIV as CaMKII is essential for LTP formation and memory functioning. CaMKIV knockout mice have spatial, emotional, and motor deficits . In rodeThe CaMKII and CaMKIV are essential kinases controlling mood and memory functions. Their dysfunction underlies various neurodegenerative and neuropsychiatric disorders. Moreover, both CaMKII and CaMKIV mediate the therapeutic effects of many central-acting drugs on the molecular level. On the basis of the reviewed literature, in order to achieve an antidepressant-like and procognitive effect, one finds that compounds should generally increase the phosphorylation of CaMKII and CaMKIV . On the The scientific findings show that the alterations in CaMKII and CaMKIV functioning are not responsible for all symptoms observed in depression, anxiety, or memory impairments. However, considering the critical role of CaMKII and CaMKIV in glutamatergic neurotransmission, which underlies many neuropsychiatric and neurodegenerative disorders, drugs targeting these kinases might help modulate some disease symptoms. Therefore, CaMKII and CaMKIV may be attractive targets for novel drugs and should be considered when designing new compounds.


Gluconacetobacter diazotrophicus. The pathway involved in the biosynthesis of bacterial EPS has not been fully elucidated, and several areas related to its molecular regulation mechanisms are still lacking. G. diazotrophicus relies heavily on EPS for survival indirectly by protecting plants from pathogen attack as well as for endophytic maintenance and adhesion in plant tissues. Here, we report that EPS from G. diazotrophicus strain Pal5 is a signal polymer that controls its own biosynthesis. EPS production depends on a bacterial tyrosine (BY) kinase (Wzc) that consists of a component that is able to phosphorylate a glycosyltranferase or to self-phosphorylate. EPS interacts with the extracellular domain of Wzc, which regulates kinase activity. In G. diazotrophicus strains that are deficient in EPS production, the Wzc is rendered inoperative by self-phosphorylation. The presence of EPS promotes the phosphorylation of a glycosyltransferase in the pathway, thus producing EPS. Wzc-mediated self-regulation is an attribute for the control of exopolysaccharide biosynthesis in G. diazotrophicus.The biosynthesis of exopolysaccharides (EPSs) is essential for endophytic bacterial colonisation in plants bacause this exopolymer both protects bacterial cells against the defence and oxidative systems of plants and acts on the plant colonisation mechanism in  The use of microbial inoculants with biofertiliser and biostimulant properties has emerged as an alternative to replace or complement the use of synthetic fertilisers to promote plant growth and development. As a result, the environmental liabilities of agricultural practices are reduced. Bio-based agricultural inputs and technologies also represent tools for increasing national sovereignty in the face of the challenges of producing food, fibres, and energy in the world.Gluconacetobacter diazotrophicus is a plant growth-promoting bacteria (PGPB) that can be found colonising the internal tissues of many plants of agricultural importance, such as sugarcane (Saccharum officinarum) ) and Walker B (hhhD). In addition, the G. diazotrophicus Pal5 Walker A (PXXGK) motif, which is important for Mg2+ binding, was located within the Wzc protein. A tyrosine cluster (YC) containing multiple tyrosine residues was located at the C-terminal end of Wzc; this tyrosine cluster is a long one (22 amino acids) and contains several tyrosine residues (seven) that constitute the self-phosphorylation sites of Wzc. In addition to these two motifs, we identified the presence of the Walker A lysine, which according to some studies, induces a strong decrease in the autokinase activity of Wzc. Finally, we identified a tyrosine G. diazotrophicus at position 585, which was able to stimulate the self-phosphorylation of the tyrosine.In wzc gene of G. diazotrophicus strain Pal5 is involved in EPS production. Therefore, unmetabolised sugar is probably responsible for a large part of this precipitate. However, it is known that mannitol (carbon source) is soluble in water–ethanol mixtures to a limited extent [Using a Tn5-mutagenesis approach, we demonstrated that the d extent . Accordid extent  and Boucd extent .E. coli [G. diazotrophicus strain Pal5 results in the inhibition of EPS biosynthesis. Here, we present evidence for a novel intercellular signalling system that controls the production of EPS by acting at the protein modification level in G. diazotrophicus Pal5, namely tyrosine phosphorylation. Our evidence indicates that the extracellular domain of the Wzc component is a receptor that directly recognizes EPS and thereby regulates kinase activity by the periplasmatic component Wzc. The EPS, as a short-range signalling molecule for cells, acts in the developing biofilm that triggers a positive feedback loop that stimulates EPS production  in G. diazotrophicus strain Pal5. Relative expression was observed when the MGD mutant grew in the presence of glucose, fructose, sucrose, and mannitol, pointing to possible autophosphorylation activity. The wzc gene expression profile results show that EPS does not regulate the expression of the wzc gene. However, EPS directs the BY kinase activity since, in the absence of EPS, the Wzc protein auto-phosphorylates, while in the presence of EPS, it phosphorylates the first glycosyltransferase in the system.Regarding the relative expression of s et al. , who obsG. diazotrophicus inhibit self-phosphorylation, thus allowing the kinase to act on other target proteins in the EPS biosynthesis pathway, such as glycosyltransferases. Therefore, EPS is commonly found in bacterial biofilms and is a regulating polymer that promotes its own synthesis. Hence, in addition to having a structural function within bacterial physiology, it would be interesting to investigate whether the EPS produced by the G. diazotrophicus Pal5 strain also has other functions in plant signalling.We experimentally demonstrated that the Wzc is rendered inoperative by self-phosphorylation and that exopolymers from G. diazotrophicus exopolysaccharide plays a critical role in several important biological processes, including adherence, interactions between bacteria and host plant cells, and resistance to host immunity. Here, we demonstrate that the self-regulation properties of Wzc from G. diazotrophicus Pal5 underscore the BY kinases-like proteins that are potentially involved in the polymerisation complex, determining the role of EPS in modulating self-phosphorylation in the biosynthesis of exopolysaccharides on the surface of G. diazotrophicus Pal5 cells. Wzc-mediated self-regulation may be a widespread feature of the exopolysaccharide production control in this endophytic diazotrophic bacterium, which is commonly associated with sugarcane plants.