Coronavirus disease 2019 (COVID-19), the illness caused by a novel coronavirus now called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to more than 260 million confirmed infections and 5 million deaths to date. While vaccination is a powerful tool to control pandemic spread, medication to relieve COVID-19-associated symptoms and alleviate disease progression especially in high-risk patients is still lacking. In this study, we explore the suitability of the rapid accelerated fibrosarcoma/mitogen-activated protein kinase/extracellular signal-regulated kinase (Raf/MEK/ERK) pathway as a druggable target in the treatment of SARS-CoV-2 infections. We find that SARS-CoV-2 transiently activates Raf/MEK/ERK signaling in the very early infection phase and that ERK1/2 knockdown limits virus replication in cell culture models. We demonstrate that ATR-002, a specific inhibitor of the upstream MEK1/2 kinases which is currently evaluated in clinical trials as an anti-influenza drug, displays strong anti-SARS-CoV-2 activity in cell lines as well as in primary air–liquid-interphase epithelial cell (ALI) cultures, with a safe and selective treatment window. We also observe that ATR-002 treatment impairs the SARS-CoV-2-induced expression of pro-inflammatory cytokines, and thus might prevent COVID-19-associated hyperinflammation, a key player in COVID-19 progression. Thus, our data suggest that the Raf/MEK/ERK signaling cascade may represent a target for therapeutic intervention strategies against SARS-CoV-2 infections and that ATR-002 is a promising candidate for further drug evaluation.The online version contains supplementary material available at 10.1007/s00018-021-04085-1. Since the emergence of the highly pathogenic and transmissible new betacoronavirus SARS-CoV-2 2019-nCoV) in Wuhan City, China, in December 2019 9-nCoV in, severe Because disease progression is dynamic, with late stages of severe COVID-19 driven by a harmful hyperinflammatory response, direct targeting antivirals (DTA) such as Remdesivir or monoclonal antibodies cannot efficiently improve health of severely diseased patients, albeit showing good effects when administered early in infection , 14.Moreover, DTA pose the risk of emerging resistant virus strains, as with the M2 ion channel inhibitor Amantadine that rapidly forced influenza virus to develop a fully resistant phenotype . In thisHuman airway epithelial cells  from ATCC were taken from the cell line collection of the IVM. The cells were grown in Dulbecco’s modified Eagle medium/Nutrient Mixture F12-Ham (DMEM/F12-HAM) (Sigma-Life Science) supplemented with 10% (v/v) fetal bovine serum (FBS) (Capricorn Scientific) and 1% (v/v) Penicillin/Streptomycin (P/S) (Sigma-Life Science).Human alveolar lung epithelial cells (A549) from ATCC, African green monkey kidney epithelial cells (VeroE6) and human embryonic kidney cells (HEK293T) from the cell line collection of the IVM were grown in Dulbecco’s modified Eagle medium (DMEM) (Sigma-Life Science) supplemented with 10% (v/v) FBS.TMPRSS2-expressing A549 cells were obtained from the German Primate Center in Goettingen. The cells were grown in DMEM supplemented with 10% (v/v) FBS, 1% (v/v) P/S, and 1 µg/ml puromycin (InvivoGen).TMPRSS2-expressing Vero76 cells were obtained from the German Primate Center in Goettingen. The cells were grown in DMEM supplemented with 10% (v/v) FBS, 1% (v/v) P/S, and 10 µg/ml blasticidin (Roth).Human ACE2-expressing A549 cells were obtained from the German Primate Center in Goettingen. The cells were grown in DMEM supplemented with 10% (v/v) FBS, 1% (v/v) P/S, 1 µg/ml puromycin, 1% (v/v) non-essential amino acid solution (NEAA) (Sigma-Life Science), and 1% (v/v) sodium pyruvate solution (Gibco).Human ACE2- and TMPRSS2-expressing A549 cells were obtained from the German Primate Center in Goettingen. The cells were grown in DMEM supplemented with 10% (v/v) FBS, 1% (v/v) P/S, 1 µg/ml puromycin, 1 µg/ml blasticidin, 1% (v/v) NEAA solution, and 1% (v/v) sodium pyruvate solution.Human colorectal adenocarcinoma cells (CaCo2) were obtained from the Department of Immunology in Tuebingen. The cells were grown in DMEM supplemented with 10% (v/v) FBS, 1% (v/v) P/S, and 1% (v/v) non-essential amino acid solution (NEAA) (Sigma-Life Science).4/ml in epithelial cell serum-free growth medium  (Thermo Fisher Scientific) plus additives [Human primary nasal AECs (Table S2) were harvested from healthy adult volunteer donors by nasal brushing of the inferior turbinate and processed for cultivation at the air–liquid interface (ALI) as described previously . Brieflydditives  supplemedditives  suppleme2 atmosphere.All cells were incubated at 37 °C and 5% COSARS-CoV-2 sputum samples were obtained from the Department of Clinical Virology of the University Hospital Muenster, after determination of the SARS-CoV-2 virus variant by sequencing. Sputum samples were centrifuged for 15 min at 4 °C and supernatants were diluted in cell culture medium, followed by filtration through a 0.5 µm syringe filter. Vero76-TMPRSS2 cells were incubated with filtered samples for 2 h at 37 °C. Afterward, cells were incubated in cell culture medium containing 2 × Antibiotic–Antimycotic (Gibco) for 96 h. Titers were determined by plaque titration. The passaging was repeated until viral concentrations were high enough to conduct experiments. Viral stocks were sequenced to exclude viral genome mutations caused by the cell culture propagation.SARS-CoV-2 viruses were handled in a laboratory approved for biosafety level (BSL) 3 work. Viruses were propagated on Vero76-TMPRSS2 cells diluted in DMEM supplemented with 2% (v/v) FBS, 1% (v/v) P/S, 1% (v/v) sodium pyruvate solution, 1% (v/v) NEAA solution, and 1% (v/v) HEPES solution (Sigma-Aldrich) (Infection-DMEM) using an MOI of 0.01. 3 days post-infection (p.i.), the supernatant was collected and the virus titer was determined by plaque titration.SARS-CoV-2 viruses (Table S1) were diluted in cell culture medium according to the desired MOI. Cells were washed twice with PBS (Sigma-Aldrich) and incubated with the viral dilutions for 1 h at 37 °C. Cells were washed once with PBS and incubated in cell culture medium.Primary AECs were washed twice with HBSS (Thermo Fisher Scientific) and incubated apically with the viral dilutions for 2 h at 37 °C. Cells were washed twice with HBSS and incubated at ALI.After the indicated total incubation times, supernatants were collected and cells were subjected to the respective analysis method.The MEK1/2-inhibitor ATR-002  (Atriva Therapeutics GmbH) was dissolved in DMSO (Roth) and used in final concentrations of 1–600 µM. DMSO served as control, using a final concentration of 0.1%.Inhibitor treatment was performed as described in the respective experiments.® 2000 (Invitrogen) according to the manufacturer’s protocol.ERK1/2 was knocked down in Calu3 cells using Lipofectamine48 h prior to infection, the cells were transfected with the siRNAs against ERK1/2 (siERK1/2) , or scrambled control siRNA (siCtrl) . 24 h post-transfection (p.t.) medium was changed.2 for 20 min.Calu3 cells were grown in 96-well flat-bottom tissue plates. 24 h after seeding cells were incubated with different inhibitor concentrations (Table S3) for 24 h or 72 h. 15 µl sterile water or 10% Triton X-100 solution (CytoSelect™ LDH Cytotoxicity Assay Kit) (Cell Biolabs Inc.) was added and incubated for 10 min at room temperature. Collected supernatants were frozen at − 80 °C until measurement. 10 µl of LDH Cytotoxicity Assay Reagent (CytoSelect™ LDH Cytotoxicity Assay Kit) was mixed with 90 µl supernatant in a clear cell culture plate suitable for a plate reader. Analysis was performed using an ELISA reader (BioTek) at 405 nm after the samples were incubated at 37 °C and 5% CO2 (Roth), and 0.01% (w/v) MgCl2 (Roth) was prepared to infect VeroE6 cells grown to a confluent monolayer in 6-well plates. 1 h.p.i. at 37 °C the virus inoculum was replaced by plaque medium composed of 63% (v/v) 2 × MEM ((20% (v/v) 10 × MEM (Gibco), 3.2% (v/v) NaHCO3 (7.5%) (Gibco), 2% (v/v) HEPES  (Sigma-Aldrich), 1.2% (v/v) BSA (35%), 1% (v/v) 100 × Penicillin/Streptomycin/l-Glutamine solution  (Gibco)), 2% (v/v) FBS, and 35% (v/v) Agar (2%) (Oxoid). Plaques were counted after 72 h incubation at 37 °C.Tenfold dilution series of SARS-CoV-2 containing solutions in PBS supplemented with 1% (v/v) P/S, 0.6% (v/v) BSA (35%) (Sigma-Aldrich), 0.01% (w/v) CaCl3VO4 (100 mM) (Sigma), and 1:200 Benzamidin (1 M) (Sigma)) centrifuged at 4 °C and 20,000 rpm for 10 min. Cleared lysates were mixed with 5 × Laemmli buffer and headed for 2 min at 95 °C. Protein separation was performed by SDS-PAGE and protein transfer onto nitrocellulose membranes by Western blotting, followed by blocking in TBS-T buffer , 0.2% Triton X-100 (Roth), 1% Tween-20 (Roth)) containing 3% BSA (w/v) (Roth) for 1 h. Membranes were incubated for 1 h or overnight with primary antibodies (Table S1) diluted in blocking buffer. A 45 min incubation with 1:3000 secondary antibody (Table S1) dilutions in TBS-T was performed to detect the primary antibodies using chemiluminescence and the Li-CorOdissey® Fc Imaging System. Signals were analyzed by the Image Studio™ (LiCor) software.Cellular and viral proteins were analyzed by Western blot. After a wash step with PBS, cells were lysed in RIPA (radioimmunoprecipitation) buffer (137 mM NaCl (Roth), 25 mM Tris–HCl (pH 8) (Roth), 2 mM EDTA (pH 8) (Roth), 10% (v/v) glycerol (Roth), 1% (v/v) NP-40 (Sigma-Aldrich), 0.5% (w/v) sodium deoxycholate (Serva), 0.1% (w/v) SDS (Roth)) supplemented with protease inhibitors (1:1000 Pefablock (200 mM) (Roth), 1:1000 Leupeptin (5 mg/ml) (Serva), 1:1000 Aprotinin (5 mg/ml) (Roth), 1:100 NaN)  antibody was diluted in blocking buffer and cells were incubated at room temperature for 1 h, prior washing and 45 min incubation with secondary antibody Alexa568-conjugated  and DAPI (5 mg/ml) . Mounting of coverslips was performed using Fluorescence Mounting Medium (Dako). The Axiovert 200 M microscope and the AxioVision V4.8.2.0 (Zeiss) software were used to capture and analyze the epifluorescence pictures.Cells on glass coverslips were fixated at 4 °C for 10 min with ice-cold methanol (Roth) (− 20 °C), washed once with PBS, and blocked in 3% (w/v) BSA in PBS at room temperature for 1 h. Monoclonal anti-SARS-CoV-2 nucleocapsid (® 480 II (Roche) in combination with the program LightCycler® 480 SW V1.5.1.62 were used for the qRT-PCR analysis. GAPDH was used as reference.Total RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. cDNA was synthesized mixing 1 µg RNA, Revert AID H Minus Reverse Transcriptase (Thermo Fisher Scientific), and oligo (dT) primers (Eurofins MWG Operon). The qRT-PCR reaction mix Brilliant III SYBR Green QPCR Master Mix (Agilent Technologies), specific primers (Table S1), and the LightCycler®) according to the manufacturer’s protocol. Cells were transfected with 0.5 µg polyI:C (InvivoGen) using Lipofectamine® 2000 (Invitrogen), followed by an incubation with different concentrations of ATR-002. 24 h.p.t. cytokine and chemokine amounts were measured using the LEGENDplex™ Human Anti-Virus Response Panel (13-plex) in combination with the Flow cytometer Gallios™ (Beckman Coulter).Cytokine and chemokine secretion was analyzed using the LEGENDplex™ Multiplex Assay (BioLegendTo quantify the levels of pERK1/2 (Thr202/Tyr204) in mock or SARS-CoV-2-infected Calu3 cells, a plate-based assay (Cisbio) was used according to the manufacturer’s protocol. Briefly, cells grown on 96-well plates  were non-infected or infected with different SARS-CoV-2 isolates for 1 h at 37 °C. Lysates were then transferred to a 384-well low-volume plate, incubated with the antibodies for 4 h at room temperature, and luminescence was recorded with a CLARIOstar reader (BMG Labtech) . HTRF ratios were normalized to maximum system output obtained through stimulation with 100 nM phorbol 12-myristate 13-acetate (PMA) (AppliChem).SARS-CoV-2 S protein receptor-mediated entry was analyzed with the SARS-CoV-2 S protein-bearing vesicular stomatitis virus (VSV) pseudotyped system, containing a G gene substitution by a green fluorescence protein gene (VSV ΔG/GFP-Luc + S Δ21). The pseudotyped virus was generated according to Berger Rentsch and Zimmer, 2011 . 24 h afCells were infected with the pseudotyped VSV ΔG/GFP-Luc + S Δ21 using an MOI of 0.003 for 1 h. ATR-002 (100 µM) treatment was initiated simultaneously with the infection (0 h) or 1 h post-infection. The solvent DMSO served as control. 16 h.p.i. GFP-positive cells were quantified by light microscopy using the Axiovert 200 M microscope (Zeiss).Western blot signal intensities were measured using the LiCor Image Studio™ version 5.2.5 (LiCor). Immunofluorescence pictures were formatted using the AxioVersion V4.8.2.0 software (Zeiss) and quantified with ImageJ 1.48v (NIH Image). LEGENDplex data were analyzed using the LEGENDplex™ Data Analysis Software version 8.0.GraphPad PRISM version 8.4.3 (GraphPad Software) was used to create the shown graphs and perform statistical analysis. Information about sample size and statistical tests are shown in the respective figure legends.We first assessed whether SARS-CoV-2 infection activated the Raf/MEK/ERK kinase cascade. Western blot analysis of SARS-CoV-2 D614G-FI)-infected Calu3 cells revealed a prominent ERK1/2 activation in the early phase of the infection, with a peak of ERK1/2 phosphorylation 1 h post-infection (h.p.i.), followed by a decrease over time  was in a range of 15.8–37.21 µM for the different SARS-CoV-2 variants including the VOCs α-B1.1.7, β-B1.351, and δ-B1.617.2 . The cytotoxic concentration that caused 50% cell death (CC50) was 895.0 µM . The resulting very favorable selectivity indices (SI) between 24.05 and 56.64 -mediated RNA interference (RNAi), with a knockdown efficacy of 70.5% ± 2.9%  were reduced by a half log unit at 50 µM ATR-002 up to 6.5 log units at 150 µM ATR-002, demonstrating a sustained inhibitory impact on the viral life cycle at non-cytotoxic concentrations . A cytopathic effect (CPE) caused by viral infection was first detected 48 h.p.i. which was not visible upon treatment with 50 µM ATR-002. Higher ATR-002 concentrations of 100 µM and 150 µM prevented CPE even 72 h.p.i. . Together, these results strongly support the suitability of the MEK1/2-inhibitor ATR-002 as a host targeting antiviral.We next addressed the question whether the antiviral activity of ATR-002 was limited to the early phase of the SARS-CoV-2 infection cycle. Therefore, cells were treated with ATR-002 either 1 h pre-infection or 2 h/4 h post-infection and the expression of viral proteins  and progeny viral titers were determined 8 h.p.i. Fig. d, e. ComTo investigate a role of the early ERK1/2 activation during the viral internalization, we used the SARS-CoV-2 S protein-bearing vesicular stomatitis virus (VSV) pseudotype system, containing a G gene substitution by the green fluorescent protein gene (VSV Δ/GFP-Luc + S Δ21) , 34. Wit. As expected, we could not detect progeny viral titers in the A549 cell lines lacking robust levels of endogenous ACE2 , whereas ACE2 overexpression allowed for a successful infection at MOI 0.01. At 48 h.p.i. and 72 h.p.i., viral titers were 3 log units and 4 log units, respectively, higher in ACE2/TMPRSS2-expressing A549 cells compared to A549-ACE2 cells. Highest viral titers were obtained in the naturally permissive Calu3 cells  and the inhibitory effect (IC50), are steady within the different virus isolates, indicating that the mutational changes of the SARS-CoV-2 variants did not influence the inhibitory ability of ATR-002. Inhibition of MEK1/2 signaling prior to SARS-CoV-2 infection was most effective, strongly arguing for an important role of the Raf/MEK/ERK pathway in the very early stage of the viral life cycle  Fig. ; Fig. S5Since hyperinflammation and multiple organ failure  , 39, 52 In conclusion, the low efficiency of DTAs against acute, self-limiting and hyperinflammatory viral diseases, such as severe influenza and COVID-19, observed so far might call for a rethinking of antiviral concepts in general. A DTA attempt might be simply too late to rescue severely diseased patients in late stages of the disease. Here, host targeting antivirals (HTA) that at the same time have immunomodulatory effects, such as ATR-002, might offer a suitable toolbox for antiviral therapies of different viral origin (different viruses misuse similar cellular factors) without posing the risk of emerging resistances. Besides promising data on bioavailability  and safeSupplementary file1 (DOCX 2872 KB)Below is the link to the electronic supplementary material.


In Parkinson’s disease, the dysfunction of the dopaminergic nigrostriatal tract involves the loss of function of dopaminergic neurons of the substantia nigra pars compacta followed by death of these neurons. The functional recovery of these neurons requires a deep knowledge of the molecules that maintain the dopaminergic phenotype during adulthood and the mechanisms that subvert their activity. Previous studies have shown that transcription factor NURR1, involved in differentiation and maintenance of the dopaminergic phenotype, is downregulated by α-synuclein (α-SYN). In this study, we provide a mechanistic explanation to this finding by connecting α-SYN-induced activation of glycogen synthase kinase-3 (GSK-3) with NURR1 phosphorylation followed by proteasomal degradation. The use of sequential deletion mutants and single point mutants of NURR1 allowed the identification of a domain comprising amino acids 123-PSSPPTPSTPS-134 that is targeted by GSK-3 and leads to subsequent ubiquitination and proteasome degradation. This study provides a detailed analysis of the regulation of NURR1 stability by phosphorylation in synucleinopathies such as Parkinson’s disease.The online version contains supplementary material available at 10.1007/s12035-021-02558-9. TH) [DAT) [AADC) [VMAT2) [NRP1 [RET (GDNF receptor) [NURR1 gene have been identified in association with Parkinson’s disease (PD), where neurodegeneration of the DAergic neurons of the SN occurs [Midbrain dopaminergic (DAergic) neurons are the main source of dopamine (DA) in the mammalian central nervous system. Several transcription factors have been implicated in DAergic differentiation . Among tTH) –7, dopamH) [DAT) , amino a) [AADC) , vesicul [VMAT2) , as well2) [NRP1  and RET eceptor) . Ablatioeceptor) –14. MutaN occurs , 16.SNCA) are associated with familiar forms of PD [DAergic neuronal loss is associated with abnormal accumulation and aggregation of the protein α-synuclein (α-SYN) in the form of Lewy bodies and Lewy neurites . Point mms of PD , 19, whims of PD , 21. Relms of PD . Howeverms of PD , 12. ThiPhosphorylation is a mechanism used in many proteins to target their proteolytic degradation. For instance, glycogen synthase kinase 3 (GSK-3) phosphorylates several proteins to create a phosphorylation-dependent degradation domain (phosphodegron) that is then recognized by a variety of E3 ubiquitin ligase adapters leading to proteasomal degradation of the phosphorylated protein . ConsideIn this study, using several models of synucleinopathy, we found that α-SYN-induced activation of GSK-3β leads to phosphorylation of NURR1 and its subsequent ubiquitin–proteasome degradation, which precedes loss of the DAergic phenotype.Human embryonic kidney 293 T with SV40 T antigen (HEK293T) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% fetal bovine serum  and 80 µg/ml gentamycin . Human neuroblastoma cells (SH-SY5Y) were cultured in RPMI supplemented with 10% fetal bovine serum (Invitrogen) and 80 µg/ml gentamicin. The SH-SY5Y-α-SYN Tet-Off cells, described previously , 38, werΔ9, pCGN-HA-GSK-3βWT, and pCGN-HA-GSK-3βY216F were provided by Dr. Akira Kikuchi . Vectors pGL3-NBRE3xLuc and pGL3-TkLuc were provided by Dr. Philippe Lefebvre . The HA-Ubiquitin expression vector was provided by Dr. Tadashi Nakagawa . The plasmid pcDNA3.1-Nurr1WT-V5/6xHis has been described previously [The vectors pCGN-HA-GSK-3βeviously . The leneviously  and SuppPurified monomeric α‐SYN was purchased from Proteos, Inc (cat no. RP‐003), and PFFs were formed according to the protocol provided by the manufacturer , 42 see.This protocol was performed as described in . Briefly3 cells/well) on poly-D-Lys-covered slides and treated with 1 µg/ml PFFs. The protocols have been previously described [129 . Secondary antibodies were as follows: Alexa Fluor 488 donkey anti-mouse, and Alexa 546 donkey anti-rabbit  and Alexa Fluor 546-conjugated donkey anti-mouse IgG . Control sections were treated identically but omitting the primary antibody.SH-SY5Y cells were seeded in 24-well plates . After 5 h, HEK293T cells were treated for 16 h with 2 μM MG132 (Sigma-Aldrich). Cells were then lysed in a RIPA buffer . Then, samples were kept for 30 min at 4 °C in a rotating wheel and centrifuged at 13,000 rpm for 10 min. Three microliters of the anti-V5 (Invitrogen) was added per lysate, and after incubation for 2 h at 4 °C in a rotating wheel, gamma-bind Sepharose-protein G was added (Amersham Biosciences), followed by incubation for 1 h at 4 °C. The complexes were harvested by centrifugation and washed three washes with RIPA buffer, resolved in SDS–polyacrylamide gels, and immunobloted. Mouse IgG TrueBlot (eBiosciences) was used as a peroxidase-conjugated secondary antibody  because it reduces interference by the 55-kDa heavy and 23-kDa light chains of the immunoprecipitation antibody.HEK293T cells co-transfected with expression plasmids for HA-Ubiquitin (HA-Ub), Nurr1WT-V5/6xHis and pCGN-HA-GSK-3βΔ9 or pCGN-HA-GSK-3βY216F using TransFectin Lipid Reagent (Bio-Rad) according to manufacturer recommendations. After 24 h of recovery from transfection, the cells were lysed in 200 μl lysis buffer . Then, the samples were sonicated and precleared by centrifugation, and 50 μl of the sample was incubated with λ-protein phosphatase  for 4 h at 37 °C. Then, the samples were resolved by SDS-PAGE and immunoblotted.HEK293T cells were co-transfected with Nurr1WT-V5/6xHis, Nurr1MUT2-V5/6xHis and pCGN-HA-GSK-3βΔ9 or pCGN-HA-GSK-3βY216F, using TransFectin Lipid Reagent (Bio-RAD) according to manufacturer recommendations. For experimental details, see HEK293T cell co-transfected with expression plasmids for Nurr1Actb and Gapdh . All PCRs were performed in triplicate.Total RNA was extracted using TRIzol reagent according to the manufacturer’s instructions (Invitrogen). Reverse transcription and quantitative PCR were done as detailed elsewhere . Primer Luciferase activities were determined using a luciferase assay system (Promega) as per the manufacturer’s instructions. As a reference plasmid to normalize transfection efficiency, a CMV-galactosidase plasmid (Promega) was cotransfected in all experiments and luciferase assay values were normalized to galactosidase activity.p ≤ 0.001), or with Student’s t test (p ≤ 0.05), using Prism version 5.03 software .Results are expressed as mean ± SEM from at least three independent experiments. Data were analyzed by one-way ANOVA followed by Newman–Keuls multiple comparison test (129-phopshorylated α-SYN (α-SYN-pSer129)  of human recombinant α-SYN . Confocal microscopy demonstrated the formation of aggregates containing α-SYN and Ser29) Fig. . PFFs inhed Fig. . By conthed Fig. . The facFFs see “”, togeth129 expression correlated with a decrease in NURR1, TH, and RET protein levels  . DOX remels Fig. . MoreoveRET Fig. . To furtter Fig. . NURR1 t9 (GSK-3β-pSer9) . Lack of Ser9 renders this kinase insensitive to downregulation by AKT-mediated phosphorylation. As a negative control, we used a hypomorphic mutant containing a single point Tyr-to-Phe mutation in its activation loop (GSK-3βY216F) that renders this kinase almost inactive [Δ9, and were not altered in the presence of inactive GSK-3βY216F. Then, we silenced the expression of both α and β isoforms of this kinase by lentiviral knock-down for 3 days in V5-NURR1 expressing HEK293T cells  Fig. . This phic genes , 50. We inactive , 52. As lls Fig.  as well lls Fig. . As a colls Fig. . Despite  Fig. . As a coΔ9, or HA-GSK-3βY216F as control. After 16 h, the cells were treated for 2 h and 4 h with the selective proteasome inhibitor MG132 (20 µM). As shown in Fig. Y216F, or HA-GSK-3βWT or HA-GSK-3βΔ9 , where X is any residue [Δ9 induced a slightly retarded EGFP-NURR1 band in the full length chimera and in the first deletion mutant (Δ1), compared to control GSK-3βY216F. In addition, the protein levels of these two constructs were decreased in the presence of GSK-3βΔ9. By contrast, the rest of the EGFP-NURR1 mutants exhibited low or no obvious band shift and were resistant to degradation in the presence of GSK-3βΔ9, suggesting that the sites targeted by GSK-3β on NURR1 are preferentially located before or at Core 2 -Pro or (Ser/Thr)-X residue . Using te 2 Fig. . As conte 2 Fig. . We foune 2 Fig. . The amie 2 Fig. .Δ9 or GSK-3βY216F  in HEK293T cells co-transfected with expression vectors for NURR1-V5 and GSK-3β16F Fig. . In the WT or NURR1MUT2 and co-transfected with GSK-3βΔ9 or GSK-3βY216F, and resolved by 2D gel electrophoresis. As shown in Fig. WT co-transfected with hypomorphic GSK-3βY216F, displayed several immunoreactive spots consistent with GSK-3β-independent posttranslational modifications of NURR1. When cells were co-transfected with active GSK-3βΔ9, we observed an increase in the intensity of acidic spots (black arrows), indicating GSK-3-mediated phosphorylation. However, in cells co-transfected with NURR1MUT2 the distribution of spots was similar in the presence of GSK-3βY216F or GSK-3βΔ9, and the acidic shift was not observed (black arrows). These results indicate that the residues of Core 2 are preferred targets of GSK-3β phosphorylation.To more precisely characterize the relevance of Core 2, HEK293T cells were transfected with NURR1MUT2. HEK293T cells were co-transfected with NURR1WT or NURR1MUT2 and GSK-3βΔ9, and exposed to CHX (100 µM)  Fig. . In the WT or NURR1MUT2. In the presence of DOX, α-SYN levels were low in cells expressing either form of NURR1, and the levels of GSK-3β-pSer9 were high, indicating its inhibition. However, in the absence of DOX, α-SYN levels increased, and GSK-3 was dephosphorylated and active. The presence of α-SYN and its downstream target, active GSK-3, completely eliminated NURR1WT protein and substantially reduced the levels of TH and RET. By contrast, the levels of NURR1MUT2 were not affected by the α-SYN/GSK-3 challenge and the levels of TH and RET remained similar to those in the absence of α-SYN.Finally, we validated the phosphorylation of Core 2 in the DAergic cell line SH-SY5Y Fig. . These c473 (inactivation) and GSK-3β-pSer9 (activation). Under these conditions, not only β-catenin but also NURR1 levels were gradually decreased. By contrast, cells co-treated with SB216763 were at least partially protected from the decrease in β-catenin and NURR1. Together, these observations confirm in a DAergic cell line the mechanistic connections between α-SYN, GSK-3β, phosphorylation, and degradation of NURR1.A highly characterized survival pathway in nerve cells is the PI3K/AKT. This pathway is activated by many growth factors and neurotrophins and leads to the inhibition of GSK-3. Therefore, we analyzed the inhibition of this pathway, by using the highly selective PI3K inhibitor LY294002. SH-SY5Y cells were submitted to a time-course of LY294002 (30 µM) alone or in combination with the GSK-3 inhibitor SB216763 (5 μM). As shown in Fig. The dysfunction of the DAergic nigrostriatal tract in PD proceeds in two phases: first, loss of the DAergic function of neurons of the SN pars compacta and then death of these neurons. The two phases can be separated by exposure to toxins that induce a transient loss of the DAergic phenotype including amphetamine, MPTP, and α-SYN , 61–65. NURR1 expression as indicated by the luciferase reporter Below is the link to the electronic supplementary material.


Specifically, ARPE-19 cells have been challenged with Aβ (1–42) oligomers, H2O2, LPS, and TNF-α, respectively. In the present study, we demonstrated that the combination of 1,25(OH)2D3 and meso-zeaxanthin significantly counteracted the cell damage induced by the three insults, at least in these in vitro integrated paradigms of AMD. These results suggest that combination of 1,25(OH)2D3 and meso-zeaxathin could be a useful approach to contrast pathological features of AMD, such as retinal inflammation and oxidative stress.Age-related macular degeneration (AMD) is a degenerative retinal disease and one of major causes of irreversible vision loss. AMD has been linked to several pathological factors, such as oxidative stress and inflammation. Moreover, Aβ (1–42) oligomers have been found in drusen, the extracellular deposits that accumulate beneath the retinal pigmented epithelium in AMD patients. Hereby, we investigated the hypothesis that treatment with 1,25(OH)  These lesions are characterized by accumulation of extracellular material, lipid, and protein aggregates. Moreover, the number and size of drusen, along with the presence of choroidal neovascularization, have been found to correlate with the stage of AMD  (Age-related macular degeneration (AMD) is a progressive neurodegenerative and multifactorial disease that if not treated or managed can impair irreversibly the visual function  in the edvanced) . Generaldvanced) . Retinaldvanced) , althougdvanced) . Currentdvanced) . Moreovedvanced) , and angdvanced) . Severaldvanced) . Furtherdvanced) . Moreovedvanced) . Macrophdvanced) . Togethedvanced) . The altin vitro model of AMD, stimulating RPE cells with three different challenges: Aβ oligomers, hydrogen peroxide (H2O2), and inflammatory stimuli (LPS and TNF-α), and testing the effects of in vitro treatment with anti-inflammatory, anti-angiogenic, and antioxidant molecules: 1,25(OH)2D3 (vitamin D3), meso-zeaxanthin (MZ), and their combination. Specifically, vitamin D3 is a secosteroid able to modulate cell differentiation, homeostasis, and apoptosis through direct and indirect mechanisms of action. The first one is activated by the binding of the active form of vitamin D3 to its receptor (VDR), a transcriptional factor. VDR is expressed in most human cells, supporting the hypothesis that vitamin D3 has a pleiotropic effect. Moreover, anti-inflammatory and anti-angiogenic effects of vitamin D3 have been widely elucidated both in in vitro and in vivo studies -b, b-carotene-3, 30-diol, MZ] is one of the three xanthophyll carotenoids localized in the d cancer . In partd cancer . A specid cancer .3 and meso-zeaxanthin activities, we tested the efficacy of these two compounds and their combination in three different in vitro models of AMD. We found that their combination significantly counteracted the damage induced by Aβ-amyloid oligomers, H2O2, and inflammatory stimuli in immortalized human RPE (ARPE-19) cells. Moreover, a bioinformatic analysis evidenced that the combination of these compounds effectively covers the pathways associated with the three stimuli, resembling the AMD multifactorial pathology.Given these premises on vitamin D2D3 , 0.1 µM of meso-zeaxanthin (MZ) , and the combination (combo) of 1,25(OH)2D3 (50 nM) and meso-zeaxanthin . Both pretreatment and treatment were performed in medium supplemented with 5% FBS to starve cells. After pretreatment, ARPE-19 cells were challenged with four different stimuli: amyloid-β oligomers  (2O2), LPS , and tumor necrosis-alpha (TNF-α) , in order to simulate retinal degeneration, retinal oxidative stress, and early and late inflammation, respectively. 1,25(OH)2D3, MZ, and the combo were also added to the medium containing negative stimuli.Human retinal pigment epithelial cells (ARPE-19) were purchased from ATCC® . Cells were cultured at 37 °C (humidified atmosphere with 5% CO2) in ATCC-formulated DMEM:F12 medium (ATCC number 30–2006) with 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal bovine serum (FBS). After reaching confluence (70%), ARPE-19 cells were pretreated for 24 h with 50 nM of 1,25(OH)42.0100) , hydroge2O2 challenge. Optimal cell density was obtained by seeding 3 × 104 cells/well in 96-well plates . After pretreatment, ARPE-19 cells were subjected to co-treatment in a fresh medium for 48 h with Aβ (1–42) (1 µM) and for 6 and 24 h with H2O2 (400 µM). At the end of the treatment, ARPE-19 cells were incubated at 37°C with MTT (0.5 mg/ml) for 3 h; then DMSO was added, and absorbance was measured at 570 nm in a plate reader . Graphs were built converting absorbance (abs) to viability (=% of control) using the following equation (absx ÷ absctrl−)  × 100, where absx is absorbance in the x well, and absctrl− is the average absorbance of negative control cells (untreated cells).The 3--2,5-diphenyl tetrasodium bromide  was used to assess cell viability after Aβ (1–42) and H4 cells/well in 96-well plates . After pretreatment, cells were co-treated for 48 h with Aβ (1–42) (1 µM) and for 6 and 24 h in the oxidative stress model (H2O2 400 µM). In control groups, only fresh medium was added. After these time points, according to manufacturer’s protocol, lysis solution was added to positive control wells (non-treated cells) for 15 min. After transferring 100 µl of medium in a new multi-well plate, 100 µl of working solution was added. After 10–15 min at room temperature, at last, 50 µl of stop solution was added. The absorbance values were measured at 490 nm using a plate reader . LDH release is reported as LDH (% control) (absx  ÷  absctrl+)  ×  100. In the equation, absx is absorbance in the x well and absctrl+ is the average absorbance of positive control cells (untreated lysed cells). Absorbance values were corrected by subtracting medium absorbance.Lactate dehydrogenase (LDH) cell release was measured using the Cytotoxicity Detection KitPLUS (LDH) . ARPE-19 cells were seeded at 2 × 103 cells/well in 96-well plates . After reaching confluence (70%), ARPE-19 cells were pretreated with 1,25(OH)2D3, mesozeaxanthin, and the combo for 24 h. Subsequently, cells were submitted to co-treatment for 48 h in amyloid-β challenge (1 µM). After treatment, media were aspirated and cells were washed by adding 100 µl/well of 1X buffer, according to manufacturer’s protocol; after washing, ARPE-19 cells were stained by adding 100 µl/well of the diluted DCDFA solution (25 µM). Cells were also incubated with this solution for 45 min at 37°C in the dark. After removing DCDFA solution, 100 µl/well of 1X buffer was added, and ROS concentration was measured immediately by detection of DCF fluorescence  with a Varioskan™ Flash Multimode Reader. According to manufacturer’s protocol, for treatment lower than or equal to 6 h, it is possible to treat cells after adding DCDFA solution. Thus, after 24 h of pretreatment with drug formulations, ARPE-19 cells were washed and stained with DCDFA for 45 min. After removing DCDFA solution and washing again, ARPE-19 cells underwent co-treatment for 6 h in H2O2 challenge (400 µM). At the end of time point, ROS concentration was measured immediately without washing. Results were reported as percentage of control after background subtraction; to determine total ROS formation, the fluorescence was normalized to the fluorescent intensity of control cells (untreated cells).ROS were measured by a 2′,7′-dichlorofluoresceindiacetate (DCFDA)–Cellular Reactive Oxygen Species Detection Assay Kit . DCFDA, a cell permeable fluorogenic dye, is deacetylated by cellular esterases to a non-fluorescent compound and later oxidized by ROS to highly fluorescent 2′,7′-dichlorofluorescein (DCF); fluorescence intensity is proportional to cell ROS concentration. Optimal cell density was obtained by seeding 20 × 10260/A280 ratio of optical density of RNA samples (measured with Multimode Reader Flash di Varioskan™) was 1.95–2.01; this RNA purity was confirmed with the electrophoresis in non-denaturing 1% agarose gel (in TAE). cDNA was synthesized from 2 µg RNA with a reverse transcription kit .Extraction of total RNA, from ARPE-19 cells, was performed with a TRIzol Reagent . The A−ΔΔCt method. Quantitative PCR experiments followed the MIQE guidelines. Gene expression levels were normalized with levels of two housekeeping genes (18S and GAPDH). Primers were purchased from Eurofins Genomics  and Qiagen . Forward and reverse primer sequences (for human genes) and the catalog number are herein listed: human IL-1β , human IL-6 , human TNF-α , human MMP-9 , human VEGF-A , human 18S , and human GAPDH .Real-time PCR was carried out with the Rotor-Gene Q (Qiagen). The amplification reaction mix included the Master Mix Qiagen (10 µl)  and cDNA . Forty-five amplification cycles were carried out for each sample. Results were analyzed with the 26. After 24 h of pretreatment with drugs and co-treatment with different stimuli , cytoplasmic and nuclear proteins were extracted by using the CER/NER kit , according to the manufacturer’s protocol. The protein content was determined by the BCA Assay Kit . Extracted proteins (20 μg) were loaded on a NuPAGE ™ 10% Bis-Tris mini protein gel . After electrophoresis, proteins were transferred to a nitrocellulose membrane . Membranes were blocked with milk, 5% Tris-buffered saline, and 0.2% Tween 20 (TBST) for 1 h at room temperature. Membranes were incubated overnight (4°C) with appropriate primary phospho-NFκB p65 , anti-GAPDH , and anti-lamin B  antibodies. After overnight incubation, the membranes were then incubated with secondary chemiluminscent antibodies  for 1 h at room temperature. After secondary antibody, the membranes were incubated with ECL  and were detected through I-Bright™ 1500  by using chemiluminescence. Densitometry analyses of blots were performed at non-saturating exposures and analyzed using ImageJ software . Values were normalized to GAPDH and lamin B, which were used as housekeeping control for cytoplasmic and nuclear fraction, respectively.ARPE-19 cells were cultured in 60-mm petri dishes at a density of 1.3 × 10in vitro model. Inputs were  β amyloid, LPS, TNF-α, H2O2, meso-zeaxanthin, vitamin D3, IL-6, Il-1β, VEGF-A, and MMP-9. The number of interactors was limited to 15, and the default confidence score was set to 0.40. Enrichment of information was included in the analysis. A centrality metrics analysis was carried out treating the network as an indirect graph , and the data are reported as mean ± SD. One-way analysis of variance (ANOVA) was carried out, and Tukey’s post hoc test was used for multiple comparisons. Differences between groups were considered statistically significant for p-values < 0.05.Statistical analysis was performed with GraphPad Prism 7 . All experiments were repeated five times , and their combination against Aβ (1–42) oligomer-induced cytotoxicity, through measurement of ARPE-19 cell viability, after challenge with Aβ 2D3 and MZ, respectively; indeed we used these concentrations also in the combination of the two compounds . 1,25(OH)2D3 and the combo pretreament significantly (p < 0.05) counteracted cell toxicity induced by challenge with Aβ  after treatment with Aβ; the tested compounds 1,25(OH)2D3 and MZ, and their combination (combo), induced a significant (p < 0.05) reduction of cell damage after 48 h (p < 0.05) ROS release in ARPE-19 cells. Only the combination of 1,25(OH)2D3 and MZ was able to significantly reduce the amount of ROS after 48 h (p < 0.05)  with Aβ . Prelimiter 48 h . Finally < 0.05) , comparep < 0.05) increased mRNA expression of IL-1β, IL-6, and TNF-α , and their combination significantly decreased IL-1β (2D3 and the combo significantly reduced TNF-α mRNA expression (p < 0.05) increased nuclear translocation of p-NFκB p65 after 48 h of insult  reduced the translocation p-NFκB p65, confirming the anti-inflammatory effect of these two compounds and their combination, in retinal pigmented epithelial cells, challenged with Aβ oligomers  significantly 2D3 (50 nM), meso-zeaxanthin (MZ) (0.1 µM), and combo 2D3 50 nM, MZ 0.1 µM), and then cells were incubated in 400 µM H2O2 for 6 h  increased LDH levels in ARPE-19 cells, and the pretreatment with tested compounds induced a significant reduction of cell damage  ROS in ARPE-19 cells, compared to control cells  counteracted oxidative stress in retinal cells, reducing ROS release.Preliminary studies on ARPE-19 cells were carried out to assess the best H for 6 h  and 24 h for 6 h . After 6l damage . Furtherol cells . Pretrea2D3 and meso-zeaxanthin (MZ) in modulation of inflammatory response, in ARPE-19 cells challenged with H2O2 (400 µM) for 6 h. H2O2 challenge led to significant (p < 0.05) increase in IL-1β and TNF-α mRNA expression (2D3 (50 nM), MZ (0.1 µM), and their combination reverted the effect of H2O2 (2O2 treatment induced a significant (p < 0.05) upregulation of both factors (p < 0.05) reduced by 1,25(OH)2D3, MZ, and their combination, compared to H2O2-treated cells   p-p65 nuclear translocation after 4 h. This process was significantly (p < 0.05) counteracted by pretreatment with 1,25(OH)2D3 (50 nM), MZ (0.1 µM), and their combination. Particularly, the combo significantly inhibited p65-NFκB translocation, compared to tested compounds and H2O2-exposed cells pression . Treatme of H2O2 . Further factors . The MMPed cells . Only th < 0.05) . Furthered cells .2D3 (50 nM), meso-zeaxanthin , and their combination 2D3 50 nM + MZ 0.1 µM) for 24h, and then exposed to LPS (150 ng/ml) for 2 h. IL-1β, IL-6, and TNF-α mRNA levels were significantly increased in the LPS-stimulated cells, compared to control cells (p < 0.05). Both compounds and their combination (p < 0.05) significantly reduced cytokine mRNA levels 2D3 + 0.1 µM MZ). Furthermore, LPS treatment significantly induced the upregulation of VEGF-A mRNA (p < 0.05) . After 2 h exposure, LPS (10 μg/ml) led to a significant increase of p-NFκB p65 nuclear translocation, in comparison to control cells (p < 0.05)  A levels . MZ sign < 0.05) , and the < 0.05) . The tre < 0.05) .p < 0.05) and were strongly downregulated by 1,25(OH)2D3, meso-zeaxanthin (MZ), and the combo pretreatments (p < 0.05) (p-NFκB p65 nuclear translocation (2D3 and MZ significantly reduced the amount of nuclear p-NFκB p65 (p < 0.05) , we analyzed TNF-α, IL-6, and IL-1β mRNA levels. After 2 h, those cytokines were significantly increased by TNF-α treatment (10 ng/ml) ( < 0.05) . We conflocation . TNF-α c < 0.05) .in vitro model of AMD through the STITCH compound app of Cytoscape v. 3.7.0, according to the approach described in the Methods section. The network was characterized by 136 nodes and 463 edges; a centrality metrics analysis was carried out treating the network as an indirect graph. Nodes with highest betweenness centrality have represented using a color scale (blue < red)  , and themyloid β , H2O2 2D3 (vitamin D3) and meso-zeaxanthin combination on several endpoints related to inflammation, oxidative stress, and cellular damage: amyloid β, H2O2, and inflammatory insults, that is, LPS and TNFα. The rationale of these integrated in vitro models of AMD is behind its multifactorial pathogenic etiology . Moreover, the combination of 1,25(OH)2D3 + meso-zeaxanthin, compared to the two single components, significantly reduced NFκB nuclear translocation, in ARPE-19 cells challenged with H2O2, LPS, and TNF-α. While in the β-amyloid model, both vitamin D3 and the combination with meso-zeaxanthin inhibited NFκB pathway activation but not the meso-zeaxanthin treatment alone.As shown by our data, vitamin D3 activity on ARPE-19 cells challenged with H2O2 and LPS are supported by recent findings on 1,25(OH)2D3 antioxidant and anti-inflammatory activity   . We provaxanthin . Additioaxanthin . Meso-zeaxanthin . Up to naxanthin . On the axanthin . Despiteaxanthin . Lack ofnt model ; this anin vitro evidence that vitamin D3 and meso-zeaxanthin association protected retinal pigmented epithelium from several damages that recapitulate the multifactorial pathogenic mechanisms of AMD. With this regard, vitamin D3 and meso-zeaxanthin supplementation would be of value in AMD patients, especially for subject diagnosed with early diagnosis of AMD, as already evidenced by several systematic reviews.In conclusion, we hereby provided


This selenated salt crystallizes as a monohydrate. In the crystal, several O—H⋯O and N—H⋯O hydrogen bonds and C–H⋯π and Se⋯O weak interactions result in a complex two-dimensional sheet-like supramolecular architecture.A hydrogen sulfate salt, [BnSeCH 15H18NSe+·HSO4−·H2O or [BnSeCH2CH(Ph)NH3+](HSO4−), of a chiral selenated amine (R)-2-(benzylselanyl)-1-phenylethanamine (BnSeCH2CH(Ph)NH2) has been synthesized and characterized by elemental analysis,1H and 13C{1H} NMR, FT–IR analysis, and single-crystal X-ray diffraction studies. The title salt crystallizes in the monohydrate form in the non-centrosymmetric monoclinic P21 space group. The cation is somewhat W shaped with the dihedral angle between the two aromatic rings being 60.9 (4)°. The carbon atom attached to the amine nitrogen atom is chiral and in the R configuration, and, the –C—C– bond of the –CH2—CH– fragment has a staggered conformation. In the crystal structure, two HSO4− anions and two water molecules form an R44(12) tetrameric type of assembly comprised of alternating HSO4− anions and water molecules via discrete D(2) O—H⋯O hydrogen bonds. This tetrameric assembly aggregates along the b-axis direction as an infinite one-dimensional tape. Adjacent tapes are interconnected via discrete D(2) N—H⋯O hydrogen bonds between the three amino hydrogen atoms of the cation sandwiched between the two tapes and the three HSO4− anions of the nearest asymmetric units, resulting in a complex two-dimensional sheet along the ab plane. The pendant arrangement of the cations is stabilized by C—H⋯π interactions between adjacent cations running as chains down the [010] axis. Secondary Se⋯O [3.1474 (4) Å] interactions are also observed in the crystal structure. A Hirshfeld surface analysis, including dnorm, shape-index and fingerprint plots of the cation, anion and solvent molecule, was carried out to confirm the presence of various interactions in the crystal structure.A hydrogen sulfate salt, C The asymmetric unit of the structure consists of one (C15H18SeN)+ cation, one HSO4− anion and a solvent water molecule with no direct hydrogen-bonding interactions between them. In the HSO4− ion, three of the S—O bond lengths are almost the same, falling in the range of 1.447 (4)–1.452 (5) Å, while the fourth is slightly elongated at 1.527 (5) Å. This suggests that the three nearly identical S—O bonds have partial double-bond character owing to resonance, while the fourth S—O bond has single-bond character. This validates the formation of the salt via single proton transfer from sulfuric acid to the amine. The title salt crystallizes in the monohydrate form in the non-centrosymmetric monoclinic P21 space group. The cation is somewhat W shaped -1-(benzylselanyl)-3-phenylpropan-2-amine  O1—H1D⋯O2, O1—H1E⋯O5 and O3—H3A⋯O1 hydrogen bonds  Å apart  along the a axis, are interconnected via discrete D(2) N1—H1A⋯O4, N1—H1B⋯O4 and N1—H1C⋯O2 hydrogen bonds , resulting in a complex two-dimensional sheet along the ab plane  interactions between adjacent cations running as chains down the [010] axis. Secondary Se1⋯O4 [3.1474 (4) Å] interactions are also observed in the crystal structure.The crystal structure features, by virtue of its salt form, several strong-to-moderate hydrogen bonds, which are not seen to the same extent in the reported freebase structure of the closely related compound  analyses of the cation, anion and the solvent are shown in Figs. 4dnorm surface of the cation , dark-red spots in the proximity of three amino hydrogen atoms are a result of strong N1—H1A⋯O4, N1—H1B⋯O4 and N1—H1C⋯O2 hydrogen bonds between the cation and HSO4− anions. Further, the Hirshfeld surface of the cation mapped over shape-index  shows a dark-red spot close to the centroid of the C1–C6 ring facing the H15 hydrogen atom, which is due to the C15—H15⋯π (π electrons of the C1–C6 ring) interactions observed between adjacent cations. The overall FP plot and those decomposed to individual atom⋯atom contacts contributing to the Hirshfeld surfaces of the cation are shown in Fig. 4c, 4d , 4e and 4f, respectively. The highest contribution to the Hirshfeld surface is from H⋯H dispersions, which contribute 48.4%, followed by C⋯H/H⋯C (26%), O⋯H/H⋯O (17.8%), Se⋯H/H⋯Se (5.7%) and others (2.1%). The symmetry about the di = de axis passing through the origin observed in the FP plots for the H⋯H and C⋯H/H⋯C contacts suggests that these interactions exist only between the cationic species and not between cation–anion or cation–water. The asymmetric nature of the FP of the O⋯H/H⋯O contacts about the di = de axis suggests that the O⋯H interactions are between unlike species, which is in agreement with the observed N—H⋯O interactions between cations and anions. A single spike observed in the FP of O⋯H/H⋯O contacts is characteristic of a strong or a moderate hydrogen bond. The spike observed at di + de ∼1.9 Å is very close to the H1C⋯O2 distance of 1.92 Å -1-(benzylselanyl)-3-phenylpropan-2-amine °, which is very similar to that observed in the title structure. No intramolecular N—H⋯Se interaction is observed in the molecular cation of the present structure, unlike in the related molecule where one is observed. In the crystal of the related amine, the molecules are linked by weak N—H⋯N interactions, generating chains along the [100] direction.The cation of the reported structure is somewhat similar to that observed in a closely related structure, (1H and 13C{1H} NMR spectra were recorded on an AVANCE-II Bruker 400 MHz spectrometer. (R)-1-(Benzylselanyl)-2-phenylethan-2-amine was synthesized according to our reported literature procedure  and used without further purification unless stated otherwise. For chemical synthesis, reactions were carried out in distilled water or in laboratory-grade solvents at room temperature. Melting points were determined in capillary tubes closed at one end and were reported uncorrected. IR spectra were recorded on a Jasco FT–IR-4100 spectrometer. Specific optical rotations (SOR) were measured on a Rudolph Autopol-I automatic polarimeter using a cell of 100 mm path length. R)-2-(benzylselanyl)-1-phenylethanamine was synthesized by a sequence of reactions shown in the reaction scheme starting from (2R)-2-amino-2-phenylethan-1-ol [derived from amino acid (R)-phenylglycinal] as per the literature procedure  in methanol under ice-cold conditions. To an ice-cold methanolic (5 mL) solution of (2R)-1-(benzylselanyl)-2-phenylethan-2-amine  was added 5 M of H2SO4 (2 mL) under stirring. The resulting precipitate was stirred for a further hour at the same temperature. Then the precipitate was filtered and washed twice with cold methanol (10 mL × 2). The white solid obtained was recrystallized from hot methanol (10 mL), which afforded colourless crystals of the title salt. The salt is soluble in water, dimethyl formamide (DMF) and dimethyl sulfoxide (DMSO), but insoluble in methanol, chloroform, dichloromethane, ether, tetrahydrofuran (THF) and hydrocarbon solvents such as n-hexane, benzene and toluene.The chiral selenated amine (c 1.0 in MeOH). Elemental analysis: found C, 46.51; H, 4.88; N, 3.54. Calculated for C15H19NO4SSe: C, 46.39; H, 4.93; N, 3.61%. FT–IR : 3452, 3027, 2925, 1615, 1537, 1361, 1186, 699, 556, 477; 1H NMR : 2.867–3.060 , 3.648 , 4.329–4.351 , 7.166–7.288 , 7.373–7.440 , 8.412 ;13C{1H} NMR : 26.99 (CH2Se), 27.15 (SeCH2), 54.75 (CH), 126.89 (C-7), 127.73 (C-13), 128.63 , 128.89 , 128.99 , 129.09 , 137.11 (C-4), 139.25 (C-10).Yield: 92%; m.p. 469–472 K; (Uiso(H) = 1.2Ueq(C) for aromatic H atoms, C—H = 0.97 Å and Uiso(H) = 1.2Ueq(C) for methylene H atoms and C—H = 0.98 Å and Uiso(H) = 1.2Ueq(C) for methine H atoms. The amino H atoms and O-bound H atoms were also positioned geometrically and refined as riding: N—H = 0.89 Å with Uiso(H) = 1.2Ueq(N); Owater—H = 0.85 Å with Uiso(H) = 1.5Ueq(Owater); Oanion—H = 0.82 Å with Uiso(H) = 1.5Ueq(Oanion).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989021010409/yy2003sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021010409/yy2003Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021010409/yy2003Isup3.cmlSupporting information file. DOI: 2114403CCDC reference:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


Scientific Reportshttps://doi.org/10.1038/s41598-020-78345-8, published online 17 December 2020Correction to: This Article contains an error in Figure Secondly, Figure 6 contains an error, where the western blotting results for α E6 (ab70), α E6 (sc-460) and α E6  are inverted black and white in panel (A) and all western blotting results are inverted black and white in panel (B). The correct Figure 6 appears below as Figure


Holt JR, Zeng W-Z, Evans EL, Woo S-H, Ma S, Abuwarda H, Loud M, Patapoutian A, Pathak MM. 2021. Spatiotemporal dynamics of PIEZO1 localization controls keratinocyte migration during wound healing . Published 27 September 2021Piezo1 GoF cells are slightly changed . For Piezo1 cKO data, while the scaling of plots is changed, the results do not change. We have corrected Figure 2 and related supplements, the related source data files, and the relevant Results description as detailed below. The main findings and conclusions of the work do not change.After publication, we became aware that there was an inadvertent error in the pixel-to-micron conversion factors used for the trajectories reported in Figure 2 and related figure supplements. We have redone the analysis using the appropriate conversion factors for Figure 2, Figure 2-figure supplement 1, Figure 2—figure supplement 2, Figure 2—figure supplement 3. Following this re-analysis, the results for The article has been corrected accordingly.Corrected Figure 2:PIEZO1 mediates speed and direction during single cell keratinocyte migration. (A) Representative differential interference contrast (DIC) images from time-lapse series of individual migrating keratinocytes isolated from ControlcKO (top) and respective Piezo1-cKO mice (bottom). Thin white lines denote the cell boundary. Scale bar = 25 µm. (B) Cell trajectories derived from tracking single keratinocytes during time-lapse experiments. Trajectories are shown with cell position at time point 0 normalized to the origin. See also Figure 2—figure supplement 1. (C) Mean squared displacement (MSD) analysis of ControlcKO and Piezo1-cKO keratinocytes tracked in B. Average MSD is plotted as a function of time. Error bars (SEM) are smaller than symbols at some points. (D) Average direction autocorrelation measurement of Piezo1-cKO and ControlcKO keratinocytes plotted as a function of time interval. * denotes a statistically significant difference, and ns denotes ‘not statistically significant’. From left to right: P = 2.0307 × 10–4, 5.75675 × 10–14, 3.18447 × 10–15, 5.34662 × 10–10, 1.72352 × 10–4, 1.34648 × 10–5, 0.01951, 0.13381, 0.61758 as determined by Kruskal-Wallis test. Plotted error bars (SEM) are smaller than symbols. (E) Quantitation of the average instantaneous speed from individual Piezo1-cKO keratinocytes relative to control cells are shown in a Cumming plot . n in B–E denotes the number of individually migrating cells tracked. See also Figure 2—figure supplements 2–3 and Figure 2 videos 1 and 2. Data are from three independent experiments from two litters. Bars in upper Cumming plots denote mean ±  s.d.Original Figure 2:PIEZO1 mediates speed and direction during single cell keratinocyte migration. (A) Representative differential interference contrast (DIC) images from time-lapse series of individual migrating keratinocytes isolated from ControlcKO (top) and respective Piezo1-cKO mice (bottom). Thin white lines denote the cell boundary. Scale bar = 25 µm. (B) Cell trajectories derived from tracking single keratinocytes during time-lapse experiments. Trajectories are shown with cell position at time point 0 normalized to the origin. See also Figure 2—figure supplement 1. (C) Mean squared displacement (MSD) analysis of ControlcKO and Piezo1-cKO keratinocytes tracked in B. Average MSD is plotted as a function of time. Error bars (SEM) are smaller than symbols at some points. (D) Average direction autocorrelation measurement of Piezo1-cKO and ControlcKO keratinocytes plotted as a function of time interval. * denotes a statistically significant difference, and ns denotes ‘not statistically significant’. From left to right: P = 2.0307 × 10–4, 5.75675 × 10–14, 3.18447 × 10–15, 5.34662 × 10–10, 1.72352 × 10–4, 1.34648 × 10–5, 0.01951, 0.13381, 0.61758 as determined by Kruskal-Wallis test. Plotted error bars (SEM) are smaller than symbols. (E) Quantitation of the average instantaneous speed from individual Piezo1-cKO keratinocytes (left) and Piezo1-GoF keratinocytes (right) relative to the respective control cells are shown in a Cumming plot . n in B–E denotes the number of individually migrating cells tracked. See also Figure 2—figure supplements 2–3 and Figure 2 videos 1 and 2. Data are from three independent experiments from two litters for conditional knockout (cKO) and six independent experiments from five litters for gain-of-function (GoF). Bars in upper Cumming plots denote mean ±  s.d.Corrected Results section:Piezo1-GoF keratinocytes also explored a somewhat larger area compared to littermate ConGoF cells, due to the cells migrating straighter with no difference in cell speed . Overall, our data demonstrate that PIEZO1 regulates keratinocyte migration, with channel knockout resulting in faster migration speed. The effects of the GoF mutation were more complex, and both Piezo1 knockout and the GoF mutation resulted in straighter trajectories.We observed that Original version:Piezo1-GoF keratinocytes and littermate ControlGoF cells . However, separating the data into directionality and speed indicated that Piezo1-GoF cells moved straighter  and slower . Overall, our data demonstrate that PIEZO1 regulates keratinocyte migration, with channel activity resulting in slower migration speed. The effects on directionality were more complex, with both PIEZO1 knockout and a GoF mutation resulting in straighter trajectories.We observed no difference in the MSD plots of Corrected Figure 2—figure supplement 1:Piezo1-cKO keratinocytes migrate further. Individual trajectories seen in Figure 2B from Piezo1-cKO (right) and ControlcKO (left) keratinocytes.Original Figure 2–figure supplement 1:Piezo1-cKO keratinocytes migrate further. Individual trajectories seen in Figure 2B from Piezo1-cKO (right) and ControlcKO (left) keratinocytes.Corrected Figure 2–figure supplement 2:Piezo1-GoF keratinocytes migrate straighter. Individual trajectories from Piezo1-GoF (bottom) and ControlGoF (top) keratinocytes.Original Figure 2–figure supplement 2:Piezo1-GoF keratinocytes migrate straighter. Individual trajectories from Piezo1-GoF (bottom) and ControlGoF (top) keratinocytes.Corrected Figure 2–figure supplement 3Single cell migration of Piezo1-GoF keratinocytes. (A) Cell trajectories derived from tracking ControlGoF (left) and Piezo1-GoF (right) keratinocytes during time-lapse experiments. Trajectories are shown with cell position at time point 0 normalized to the origin. (B) Mean squared displacement (MSD) analysis of ControlGoF and Piezo1-GoF keratinocytes tracked in A. Average MSD is plotted as a function of time. Error bars (SEM) are smaller than symbols at some points. From left to right, asterisks denote P = 0.04647, 0.04424, 0.03956 as determined by Kruskal-Wallis test. (C) Quantitation of the average instantaneous speed from individual Piezo1-GoF keratinocytes relative to control cells are shown in a Cumming plot . (D) Average direction autocorrelation for Piezo1-GoF and ControlGoF keratinocytes plotted as a function of time interval. * denotes a statistically significant difference, and ns denotes ‘not statistically significant’. From left to right: P = 7.66613 × 10–4, 3.22610 × 10–1, 8.00140 × 10–1, 1.26680 × 10–1, 2.80100 × 10–2, 1.17235 × 10–4, 1.70078 × 10–8, 2.47432 × 10–8, 1.76724 × 10–14, 4.01667 × 10–13, 2.97488 × 10–16, 3.54287 × 10–15, 4.08085 × 10–12, 3.60513 × 10–12, 9.64395 × 10–11, 3.83225 × 10–10, 1.31866 × 10–8, 7.45673 × 10–8, 9.63184 × 10–5, and 4.16131 × 10–5 as determined by Kruskal-Wallis test. Related to Figure 2. Data are from six independent experiments from five litters. Data in B and D are presented as the mean ± SEM. See also Figure 2—video 2.Original Figure 2–figure supplement 3Single cell migration of Piezo1-GoF keratinocytes. (A) Cell trajectories derived from tracking ControlGoF(left) and Piezo1-GoF (right) keratinocytes during time-lapse experiments. Trajectories are shown with cell position at time point 0 normalized to the origin. (B) Mean squared displacement (MSD) analysis of ControlGoF and Piezo1-GoF keratinocytes tracked in A. Average MSD is plotted as a function of time. Error bars (SEM) are smaller than symbols at some points. (C) Average direction autocorrelation for Piezo1-GoF and ControlGoF keratinocytes plotted as a function of time interval. * denotes a statistically significant difference, and ns denotes ‘not statistically significant’. From left to right: P = 7.922194 × 10–4, 0.3263, 0.8208, 0.14186, 0.02523, 1.03604 × 10–4, 1.42496 × 10–8, 2.18161 × 10–8, 1.71116 × 10–14, 3.13349 × 10–13, 3.0169 × 10–16, 2.17203 × 10–15, 3.3468 × 10–12, 2.83094 × 10–12, 1.17488 × 10–10, 3.54255 × 10–10, 1.21566 × 10–8, 7.6758 × 10–8, 8.58726 × 10–5, 3.76797 × 10–5 as determined by Kruskal-Wallis test. Related to Figure 2. Data are from six independent experiments from five litters. Data in B and C are presented as the mean ± SEM. See also Figure 2—video 2.


Fortuynia churaumi, F. shibai, F. rotunda and Alismobates reticulatus are described and details about juvenile morphology of the selenoribatid species, Arotrobates granulatus, Indopacifica taiyo and I. tyida are given. Juveniles of the genus Arotrobates are reported for the first time and strong ventral femoral carinae are supposed to be diagnostic characters. Basically, the conservative morphology of immature stages could be confirmed in every studied taxon and its importance for phylogenetic considerations is emphasized.The present paper provides information on juvenile stages of seven intertidal oribatid mite species from the East Asian region. Larva and nymphs of the fortuyniid species  Much of the life of oribatid mites is spent as a juvenile and therefore knowledge about these active developmental stages is important for reasons relating to ecology and systematics Travé . GrandjeThalassozetes riparius Schuster, The intertidal oribatid mite families Fortuyniidae and Selenoribatidae represent two of the few exceptions, as juvenile morphology played an important role in early systematic considerations. Moreover, numerous species descriptions provided additional information on immatures resulting in a considerable amount of knowledge about juvenile morphology. Van der Hammen  erected Circellobates venustus, where Luxton  for morphological investigation. Most samples were collected by the authors, and different collectors are given next to the respective sample. Juveniles were assigned to a specific species only if adults of a single species were contained in the respective sample.Fortuynia churaumi – (1) Ishigaki-jima, pref. Okinawa: Yamabare, thick cushions of Bostrychia on large rocks; 24°26ʹ56.11”N 124°10ʹ46.41”E; 20 March 2019. (2) Okinawa-jima, pref. Okinawa: Sosu, Bostrychia in large crack on huge single rock; 26°48ʹ30.23”N 128°19ʹ6.00”E; 22 March 2019.Fortuynia shibai – (1) Omi-shima, pref. Ehime: black intertidal algae on mussels and barnacles on concrete wall; 34°13ʹ26.60”N 132°59ʹ0.68”E; 17 September 2018. (2) Kii-hantō, pref. Wakayama: Shirahama-cho, Nishinmuro-gun, Seto Marine Biological Laboratory, Field Science Education and Research Center; 33°41ʹ32”N 135°20ʹ04”E; 18 June 2019.Fortuynia rotunda – Iriomote-jima, pref. Okinawa: Estuary of Goyoshi river, brown algae on mangrove floor and roots, 24°19ʹ17”N 123°54ʹ38”E; 16 March 2019.Alismobates reticulatus – (1) Iriomote-jima, pref. Okinawa: Estuary of Goyoshi river, brown filamentous algae on mangrove floor, 24°19ʹ17.43”N 123°54ʹ38.87”E; 16 March 2019. Nadara river estuary, Bostrychia and other algae from boulder; 24°23ʹ52.93”N 123°49ʹ42.72”E; 16 March 2019. (2) Yonaguni-jima, pref. Okinawa: Kataburu Beach, Bostrychia and smaller plants on small rocks; 24°26ʹ25.78”N 122°58ʹ27.44”E; 18 March 2019. (3) Ishigaki-jima, pref. Okinawa: Kabira Bay, algae on rocks; 24°26ʹ56.2”N 124°08ʹ47.3”E; 31 March 2019; leg. H. Uchida.Alismobates granulatus – (1) Amami-oshima, pref. Kagoshima: Koniya Port, Bostrychia from small crevices on rough boulder; 28° 8ʹ51.19”N 129°18ʹ18.99”E; 24 March 2019. (2) Ishigaki-jima, pref. Okinawa: Kabira, unknown algae on rocks; 24°28ʹ25.7”N 124°08ʹ10.5”E; 31 March 2019; leg. H. Uchida.Indopacifica taiyo – (1) Yonaguni-jima, pref. Okinawa: Kataburu Beach, Bostrychia on cliffs; 24°26ʹ19.18”N 122°58ʹ21.81”E; 18 March 2019. (2) Amami-oshima, pref. Kagoshima: Isu Bay, Bostrychia on large boulder; 28°10ʹ12.79”N 129°21ʹ34.81”E; 24 March 2019.Indopacifica tyida – (1) Yonaguni-jima, pref. Okinawa: Sonai, dark black filamentous algae on rocks; 24°28ʹ15.69”N 122°59ʹ55.38”E; 19 March 2019.https://inkscape.org) and finally they were further modified with Adobe Photoshop CS.Preserved specimens were embedded in Berlese mountant for microscopic analysis in transmitted light. Drawings were performed with an Olympus BH-2 Microscope equipped with a drawing attachment, then they were scanned and afterwards processed and digitized with the free and open-source vector graphics editor Inkscape  thin, long, lamellar seta (le) also thin but slightly shorter. Minute or alveolar exobothridial (ex) and interlamellar seta (in). Bothridium small cup-like and laterally opened. Sensillum short, clavate. Gnathosoma no obvious differences from adult stage. Gastronotum oval in dorsal view, slightly convex in lateral view. Posterior half of centrodorsal plate with lesser sclerotized area resembling inverted Y, present in nymphs. Large folds framing centrodorsal plate, showing fine granular surface. Orifice of opisthonotal gland gla located in posterior third of lateral folds. Legs with paraxial porous areas present on all femora.Apheredermous. Colour dark brown. Integument plicate and soft, except for centrodorsal plate. Prodorsum triangular, rostrum rounded, cerotegument overall finely granular. Rostral seta : length 200–234 µm (mean 224 µm).Prodorsum ). Interl1-3, da, dm, dp, la, lm, lp, h1-2c, all distally slightly serrate, except for h2. Transversal ridge on centrodorsal plate passing posterior line of setae lm and dm.Gastronotic region ). ElevenPodosoma and venter ). EpimerLegs . Femora (N = 10): length 277–313 (mean 292 µm).1-3, da, dm, dp, la, lm, lp, h1-3, p1-3c. Seta 1, da, dm, la, lm, h2c long (43–62 µm) and distally slightly barbed, 2, c3, lpc spiniform normal (22–31 µm), 3dp, h short (ca. 8 µm) and 1h very short (ca. 4 µm). Setae 1-3p spiniform, normal length (20–30 µm).Gastronotic region . Fifteen1 c and 4a added in this stage. One pair of short genital setae 1g. Adanal and anal setae not developed. Cupule of lyrifissure ip anterior of anal opening.Podosoma and venter ). EpimerLegs . Porous (N = 14): length 344–406 µm (mean 364 µm).1-3p positioned more laterally, 1p longest (102–114 µm), 2-3p short (11–13 µm).Gastronotic region ). Fiftee4b added in this stage. Two pairs of short genital setae 1-2g. One pair of aggenital setae. Three pairs of adanal setae flanking anal orifice. Two pairs of vestigial anal setae. Cupule of lyrifissure iad anterior of anal opening.Podosoma and venter ). EpimerLegs . Porous (N = 4): length 400–463 µm (mean 429 µm).Gastronotic region ). Fiftee3 c added in this stage, close to trochanter III. Four pairs of genital setae 1-4g. Three pairs of adanal setae; 2-3ad short (ca. 9 µm), 1ad at least twice as long (ca. 40 µm). Two pairs of short anal setae (9–11 µm), fully developed in this stage.Podosoma and venter ). EpimerLegs. Setation and solenidia are shown in F. shibai is already present in the larval stage and remains constant throughout the development. Fortuynia shibai adults also show porous areas on all femora and trochanters III and IV. The femoral porous areas are already present in all juvenile stages but porous areas on trochanters could not be confirmed in any ontogenetic stage (either they are difficult to trace or they are only developed in the adult stage).The barbulation of notogastral setae shown in the adults of Fortuynia churaumi Pfingstl, Shimano & Hiruta, 2019Larva and protonymph unknown.(N = 1): length 350 µm.Prodorsum. Interlamellar seta and exobothridial setae minute.1-3, da, dm, dp, la, lm, lp, h1-3, p1-3c. All setae approx. same length (19–22 µm), except for 2h considerably longer (31 µm).Gastronotic region. Fifteen pairs of normal spiniform notogastral setae; 4b added in this stage. Two pairs of short genital setae 1-2g (13–16 µm). One pair of aggenital setae. Three pairs of adanal setae flanking anal orifice. Two pairs of vestigial anal setae.Podosoma and venter ). EpimerLegs. Setation and solenidia are shown in (N = 16): length 388–419 (mean 396 µm).Gastronotic region . Fifteen3 c close to trochanter III. Four pairs of genital setae 1-4g. One pair of aggenital setae. Three pairs of adanal setae, 1ad longest (ca. 28 µm). Two pairs of anal setae fully developed.Podosoma and venter ). EpimerLegs . Femora Fortuynia churaumi adults show a specific reticulate pattern (fine dense granulation interrupted by non-granular lines) on their femora  and these setae are considerably longer than in F. churaumi.Fortuynia rotunda Marshall & Pugh, 2002(N = 1): length 262 µm.Prodorsum (7B). Interlamellar and exobothridial seta minute.1-3, da, dm, dp, la, lm, lp, h1-2c. Anterior notogastral setae (50–55 µm) three times longer than posterior notogastral setae (12–15 µm). Transversal ridge on centrodorsal plate passing posterior line of setae lm and dm medially incomplete.Gastronotic region ). ElevenPodosoma and venter. Epimeral setation 2-1-2. Claparède’s organ globular, nearly completely covered by epimeral cuticle. Aggenital, genital, adanal and anal setae not developed.Legs. Setation and solenidia see (N = 1): length 350 µm.1-3, da, dm, dp, la, lm, lp, h1-3, p1-3c. Anterior notogastral setae (65–70 µm) approx. twice as long as posterior notogastral setae (25–30 µm).Gastronotic region. Fifteen pairs of simple notogastral setae; Podosoma and venter ). EpimerLegs. Setation and solenidia are shown in (N = 1): length 544 µm.Gastronotic region ). Fiftee3 c added in this stage, close to trochanter III. Four pairs of genital setae. Three pairs of adanal setae. Two pairs of anal setae, fully developed in this stage.Podosoma and venter. Epimeral setation 3-1-3-2, seta Legs. Large porous areas on paraxial side of each femur. Porous areas on trochanters not traceable. Setation and solenidia see Fortuynia rotunda from African coasts, they gave short information on the legs, e.g. colour and length of segments, and only details about the setae of genu I and II . Karasa p. 179,  providedFortuynia rotunda is characterized by its very rounded notogaster and the juveniles already show a similar body shape with the gastronotic region being considerably broad and rounded. However, this trait should not be considered diagnostic, neither for adults nor for juveniles because it is also present in all developmental stages of F. smiti  located in posterior third of lateral folds. Ventral sejugal suture developed as distinct furrow, posterior border medially opened over a short distance. Legs monodactylous with large hook-like claws. Large porous areas on same leg segment as in adults.Apheredermous. Colour dark brown. Integument plicate and soft, except for centrodorsal plate. Prodorsum triangular, anterior part finely granular, rostrum rounded. Rostral and lamellar seta short and simple. Minute exobothridial and interlamellar setaAlismobates reticulatus Luxton, 1992Larva and protonymph unknown.(N = 1): length 222 µm.1-3, da, dm, dp, la, lm, lp, h1-3, p1-3c.Gastronotic region. Large centrodorsal plate showing reticulate surface pattern, except for inverted Y-shaped area on posterior third of gastronotic region. Fifteen pairs of short spiniform notogastral setae (8–10 µm); 1-3ad flanking anal orifice. Two pairs of vestigial anal setae.Podosoma and venter. Epimeral setation 3-1-2-1. Two pairs of short genital setae (5–6 µm). One pair of aggenital setae. Three pairs of adanal setae Legs. Setation and solenidia are shown in (N = 4): length 262–283 (mean 276 µm).Gastronotic region ). Fiftee4b close to trochanter IV. Three pairs of genital setae 1-3g. One pair of aggenital setae. Three pairs of adanal. Two pairs of anal setae, fully developed.Podosoma and venter ). EpimerLegs. Setation and solenidia see A. reticulatus are characterized by the name giving reticulate cuticular notogastral pattern, whereas this pattern is best observed around the medial notogastral setae : 243–265 (mean 254 μm).1-3, da, dm, dp, la, lm, lp, h1-3, p1-3c.Gastronotic region. Fifteen pairs of normal spiniform notogastral setae (8–12 µm); 1an).Podosoma and venter. Epimeral setation 1-0-1-1. Two pairs of short genital setae (ca. 5 µm) arranged in longitudinal row. Three pairs of vestigial adanal setae flanking anal valves. One pair of vestigial anal setae (Legs . SetatioLength (N = 5): 274–299 (mean 285 μm).Gastronotic region ). No difPodosoma and venter ). Epimer1-3g (4–5 µm) in longitudinal row. Three pairs of vestigial adanal setae. Anal setae 1an developed in this stage.Three pairs of short genital setae Legs. Setation and solenidia are shown in Arotrobates, he provided sketchy drawings of the legs of A. lanceolatus and mentioned at least in the key that members of this genus show conspicuous crests on all femora. Apart from this info, he provided very few further details on leg features whereas he mostly reported appearances of solenidia, for both known species. Later, Karasawa and Aoki : 194 μm (both the same size).1-3, da, dm, dp, la, lm, lp, h1-3c and 1-3p, all short, approximately same length (8–10 µm).Gastronotic region. Fifteen pairs of notogastral setae; 4a medially on epimeron IV. One pair of short genital setae. Aggenital setae absent.Podosoma and venter. Epimeral setation 1-0-1-1. Seta Legs. Claws with one proximoventral tooth. Setation and solenidia see Length (N = 5): 206–237 (mean 227 μm).Gastronotic region ). Fiftee1-2ad flanking anal valves. Two pairs of vestigial anal setae.Podosoma and venter. Epimeral setation 1-0-1-1. Two pairs of short genital setae (approx. 4 µm) arranged in a longitudinal row. Two pairs of adanal setae Legs. Claws with one proximoventral tooth. Setation and solenidia are shown in Tritonymph unknown.Indopacifica tyida Pfingstl, Shimano & Hiruta, 2021Larva and protonymph unknown.Length (N = 3): 250–295 (mean 276 μm).1-3, da, dm, dp, la, lm, lp, h1-3c and 1-3p, all short, approximately same length (6–10 µm).Gastronotic region ). Fiftee1-2ad (ca. 3 µm) flanking anal valves. Two pairs of vestigial anal setae.Podosoma and venter. Epimeral setation 1-0-1-1. Two pairs of short genital setae (ca. 4 µm) arranged in longitudinal row. Two pairs of short adanal setae Legs . Claws wLength (N = 1): 325 μm.Gastronotic region ). No dif1-2an (4 µm).Podosoma and venter. Epimeral setation 1-0-1-1. Three pairs of short genital setae (4 µm) in longitudinal row. Two pairs of adanal setae and two pairs of short anal setae Legs. Claws with one proximoventral tooth. Setation and solenidia are shown in Indopacifica species are more or less identical in terms of habitus, prodorsal, notogastral and ventral setation. The only found differences between I. taiyo and I. tyida juveniles concern the development of tibial setae, i.e. the antiaxial lateral setae on tibia I and III appear already in the deutonymphal stage in I. tyida but they appear later (possibly in tritonymph but due to lacking data not confirmed yet) in I. taiyo.Juveniles of the Japanese Psednobates, Rhizophobates, Thasecazetes.Grandjean  organizeAlismobates reticulatus and A. pseudoreticulatus show a distinct reticulate pattern on their centrodorsal plate and this pattern is also present in the adults. All other known Alismobates juveniles show a clear foveate pattern that is absent in the adult stage. Consequently, A. reticulatus and A. pseudoreticulatus may share a common ancestor and the reticulate pattern may represent a synapomorphy of these two species. Alismobates reticulatus is distributed from Hong Kong , which would allow distinction at least in the deutonymphal stage. Basically, all Indopacifica juveniles show a strongly granular cerotegument, a feature that is only similarly shown in A. granulatus nymphs, and they all possess exobothridial setae that are shaped like a minute lamella. The latter trait may be diagnostic for the juveniles of this genus but such tiny structures are often misinterpreted in light microscopic view and thus need further confirmation with other means of observation.Similar to


The shortest interaction, 2.53 (2) Å, involves the hydridic hydrogen H01, which fulfils a bridging function in the formation of chains of KHBEt3 units parallel to the a axis .In the title compound, formally K If the neighbouring asymmetric units generated by the 21 screw axis parallel to the a axis (see next section) are considered, there are a total of eleven K1—H distances shorter than 3 Å, with no clear limit as to what might be considered a ‘bonding’ distance. One further such distance involves the 21 screw axis parallel to the c axis. The environment of the potassium atom is shown in Fig. 2cf. the ionic formulation of the title compound in Table 2ca 2.8–2.9 Å, involving methyl hydrogen atoms, have been postulated as structurally significant in a TMEDA complex of potassium diisopropylamide  Å, H01—K1—H01i = 104.0 (4)°, K1—H01—K1ii = 126.7 (9)°. The distance between adjacent potassium atoms in the chain is 4.6839 (6) Å.To a first approximation, ignoring all interactions at K1 except for K1—H01, the molecules are connected by the appropriate 2is Fig. 3. The hydConQuest Et3BH]2 , but with 1,3,5-trimethyl-1,3,5-triazanonane, was reported by Krieck et al. La(thf)}2(μ-Cl)]  in n-hexane. The standard work-up procedure included removal of the solvent under dynamic vacuum, extraction of the residue with n-hexane and filtration. The filtrate was concentrated and cooled to 243 K. After several days, a few pale-yellow crystals were harvested. However, in contrast to our expectations, these did not consist of [{(η5:κ-N-pdl*SiMe2NtBu)La(thf)}2(μ-H)], but of the starting reagent KHBEt3.We attempted the preparation of a rare-earth metal hydride by salt metathesis between [{(ηUiso(H) values were fixed at 1.2 (for methylene groups) or 1.5 (for methyl groups) times the equivalent Ueq value of the parent carbon atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989021004734/yz2007sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989021004734/yz2007Isup2.hklStructure factors: contains datablock(s) I. DOI: 2081809CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


J Clin Exp Dent. 2021 Apr1;13(4):e418-21), there is an error in the authors of the manuscript. Thecorrect author list is: Pablo Garrido-Martínez, Juan-Francisco Peña-Cardelles, NorbertoQuispe-López, José-Juan Pozo-Kreilinger, Germán Esparza-Gómez, Néstor Montesdeoca-García,José-Luis Cebrián-Carretero.In this article by Garrido-Martínez P and colleagues (


We have shown previously, that the 18 kDa translocator protein (TSPO) synthetic ligands quinazoline derivatives (2-Cl-MGV-1 and MGV-1) can inhibit activation of in BV-2 microglial cells. In the present study we assessed the impact of novel TSPO ligands on lipopolysaccharide (LPS)-induced microglial activation as expressed by release of pro-inflammatory molecules, including cytokines  nitric oxide (NO), CD8, and cyclo-oxygenase-2 (COX-2). The TSPO ligands 2,4-Di-Cl-MGV-1, CB86, and CB204 counteracted with the LPS-induced microglial activation. Exposure to LPS along with the TSPO ligand 2,4-Di-Cl-MGV-1 (25 μM) reduced significantly the release of NO by 24-, IL-6 by 14-, IL-β by 14-, IFN- γ by 6-, and TNF-α by 29-folds, respectively. In contrast to the anti-neuroinflammatory effect of the TSPO ligands, the effect of diclofenac sodium  did not reach statistical significance. No alterations in IL-10 and IL-13 were detected (M2 anti-inflammatory pathway) during the inhibition of M1 pro-inflammatory pathway. TSPO isThe microglial cells are the brain’s resident macrophages and are involved in innate-inflammatory responses in the central nervous system (CNS) . M0, arePenetration of infectious agents via the blood brain barrier (BBB) into the brain, leads to microglial activation which eventually results in the secretion of pro-inflammatory cytokines including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ .Microglial toll-like receptors (TLR)-4 are activated by exposure to lipopolysaccharide (LPS), an endotoxin produced by the cell wall of gram-negative bacteria . The stiActivation of the NF-κB transcription factor leads to a marked macrophage-dependent immune response . These cLocal effects of inflammation are usually beneficial, for example acute destruction of invading microorganism. It is also well known that chronic inflammatory response can have significant harmful effects . Anti-inMicroglia are pivotal players in innate immune/inflammatory responses in multiple neurologic disorders, including Alzheimer Disease , ParkinsIn a recent study, we demonstrated the anti-inflammatory effects of the TSPO ligands 2-Cl-MGV-1 and MGV-1 .The aim of the present study was to evaluate the anti-inflammatory activity of the novel TSPO ligands 2,4-Di-Cl-MGV-1, CB86, and CB204 in BV-2 microglial cell line activated by LPS. To this end we assessed the inhibitory effect of the ligands on LPS-induced inflammatory response in BV-2 cells, as reflected by cell viability, metabolic activity, nitric oxide (NO) production, release of pro-inflammatory cytokines , and the expression of CD8, cyclo-oxygenase-2 (COX-2) and NF-κB p65. Diclofenac sodium  was used as an anti-inflammatory comparator. We expected that these ligands would exhibit better anti-inflammatory activity compared to the previously studied two TSPO ligands (2-Cl-MGV-1 and MGV-1) due to a better affinity to TSPO.22.12.2Procedure: To a suspension of potassium hydride (6.75 mmol) under argon in 30 ml of 1,2-dimethoxyethane (DME) is added the 2-quinazolin-4-ol (A) (4.5 mmol) in one portion. The reaction mixture was stirred for 30 min at room temperature and the carbamoyl chloride (B)  was added dropwise to the reaction mixture. Reaction mixture was then brought to reflux for 5 h till completion of reaction (TLC monitored). The reaction mixture was then quenched with 30 mL of water carefully, followed by extraction with dichloromethane (3 × 50 mL). Combined organic phases were dried over MgSO4 and the solvents were evaporated under reduced pressure. The resulting crude product was purified by silica gel chromatography using hexane/EtOAc as eluent (85/15).2.3The two compounds CB86 and CB204 were synthesized and apparently, the affinity of CB86 is better than the classical TSPO ligand PK 11195 .Image 32.4in-vitro model of M1 inflammatory pathway using murine BV-2 cells, derived from raf/myc-immortalized murine neonatal microglia . These cells were found to be appropriate for pharmacological characterization, including immunological functions. These cells can be activated by LPS or IFN-γ , and streptomycin (100 μg/ml) (These BV-2 microglia cells were cultured at 37 °C in an atmosphere of 5% CO μg/ml) .2.5BV-2 cells were seeded in 6-wells plate. After 48 h, the cells were exposed to 100 ng/ml LPS  for 24 h, as previously described . Medium 2.6Treatments included: 24-h exposure of BV-2 cells to LPS (100 ng/ml), with or without the treatment of 2,4-Di-Cl-MGV-1 and DS (25 μM of each). In a dose-response experiment of 2,4-Di-Cl-MGV-1, it was shown that already at 1 μM 2,4-Di-Cl-MGV-1 has a significant inhibitory effect on microglial activation but, the effect of 10 μM of 2,4-Di-Cl-MGV-1 was significantly higher than control. The 25 μM concentration was found to be optimal for restoration of the inflammatory markers to control levels . Therefo2.7This method was used to count the viable cells in order to seed a same number of cells for the various groups of each experiment. Viable cells exclude the Trypan blue dye, whereas nonviable cells absorb the dye and appear blue. The assay was performed as previously described . Briefly2.8We used the cell metabolic-XTT-based assay kit , to assess the metabolic activity of BV-2 cells exposed to LPS. According to the instructions of the manufacturer, briefly, the 2,3-bis [2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxyanilide inner salt (XTT) assay is based on reduction of XTT by mitochondrial dehydrogenases of viable cells yielding an orange formazan product. Absorbance at 492 nm with reference at 620 nm was measured with the Spectrophotometer Zenyth 200  .2.9Nitrite and nitrate are the two stable forms of nitric oxide (NO) which are released during inflammatory responses by iNOS. BV-2 cells were seeded in 24 well plate and incubated for 72 h in complete medium. Then, the cells were exposed to 100 ng/ml of LPS and treated with 2,4-Di-Cl-MGV-1 or DS (25 μM). Nitrite production was determined by measuring the levels of the NO metabolite nitrite (NO2) in the medium by using a colorimetric reaction with Griess reagent (explained in 2.10ELISA kits were used for the assessment of the expression of IL-1β (ab197742), TNF-α (ab208348), IFN-γ (ab100689), IL-6 (ab222503), IL-10 (ab108870), and IL-13 (ab219634) in the cell culture supernatant and NF-κB p65 (ab176647) and COX-2 (ab210574) in cell lysates . All the detected markers were compared between LPS-exposed cells and LPS + ligands-treated cells. All the reagents provided in the kit were ready for use and stored according to the manufacturer’s instructions.Sample preparation: Cell culture supernatants were collected and centrifuged at 210 g to remove floating cells. Cell culture supernatants were carefully collected, aliquoted and stored in −20 °C. For lysate samples, growth medium was removed by aspiration with vacuum, and cells were washed 2 times in PBS. Cells were scraped off and resuspended by the addition of chilled 1X Cell Extraction Buffer PTR (to lyse the cells with 100 μL of 1X Cell Extraction Buffer PTR). Cells were incubated on ice for 20 min and then centrifuged at 15,300 g for 5 min at 4 °C. Supernatant was collected, aliquoted, and stored in −80 °C immediately.Plate preparation and standards: The kits  provided ready to use plates and desired number of wells were used for the experiment. 50 μL of all samples, controls and standard were added to the appropriate wells. 50 μL of the antibody cocktail was added to the samples in each well. The wells were sealed and incubated for 1 h at room temperature on a plate shaker set to 400 rpm. Plate was washed with 3 × 350 μL 1X Wash Buffer PT. TMB, 100 μL substrate was added and incubated for 15 min in the dark on a plate shaker set to 400 rpm 100 μL of stop solution was added and mixed on a shaker for 1 min to stop the reaction. O.D. was recorded the at 450 nm with endpoint reading.2.11g at 4 °C for 20 min. The pellet was resuspended in around 50 ml of 50 mM ice cold phosphate buffer, pH 7.4 to achieve 750 μg/ml protein. The protein concentration was determined using Bradford assay.Rat kidney membranes were used in the binding assays since it expresses the TSPO and is an established tissue for the binding characteristics of TSPO. Sprague Dawley rats were anesthetized with ketamine (87.5 mg/kg) and xylazine (12.5 mg/kg) cocktail that was injected IP. The rats were sacrificed by decapitation. The kidneys were collected and homogenized for 30 s using a Brinkmann Polytron (setting 10) in 50 mM phosphate buffer, pH 7.4. The homogenate was centrifuged at 49,000×2.123H] PK 11195  binding to kidney membranes was assessed. The binding assay in final volume of 500 μL containing 25 μL of [3H] PK 11195  in the absence  or the presence of various concentrations of unlabeled ligands  and 400 μL of kidney membranes (300 μg protein). The mixture was incubated for 90 min on ice. Samples were filtered through Whattman GFC filters under vacuum and washed three times with 50 mM phosphate buffer. Filters were placed in vials containing 3 ml of liquid scintillation (LSC) cocktail. The samples were counted for radioactivity after 12 h.The ability of unlabeled PK 11195, MGV-1, 2-Cl-MGV-1, and 2,4-Di-Cl-MGV-1 to compete with  PK 11195  binding to kidney membranes  is one of the inflammatory markers but under aerobic conditions, it spontaneously oxidizes to its stable forms i.e. nitrite and nitrate. So, nitrite levels were analyzed as an inflammatory marker. BV-2 cells were exposed to 100 ng/ml of LPS along with 25 μM of 2,4-Di-Cl-MGV-1, CB86, CB204 or DS for 24 h. Nitrite level in the LPS group was dramatically elevated but in the case of LPS+2,4-Di-Cl-MGV-1 and LPS + CB86, induction of nitrite was prevented completely (around 24-folds). In contrast, DS did not prevent significantly the induction of nitrite by LPS and CB204 has some effect for the inhibition of nitrite levels, but it is not as good as 2,4-Di-Cl-MGV-1 and CB86 . All theAlso, there was no significant difference between naïve, 1% ethanol vehicle and 1% DMSO vehicle. Thus, we used 1% ethanol in the case of 2,4-Di-Cl-MGV-1 and DS; whereas 1% DMSO as a control in the case of CB86 and CB204.3.3The effects of 2,4-Di-Cl-MGV-1 on IL-1β, TNF-α, IFN-γ, and IL-6 generation as a part of pro-inflammatory response of BV-2 cells induced by LPS were assessed using ELISA A–D. No d3.4We conducted an NF-κB p65 (pS536) ELISA assay in order to detect NF-κB p65 activation. As seen in 3.5COX-2 is an intracellular marker of the inflammatory pathway. Therefore, we conducted COX-2 ELISA assay. As shown in 3.6CD8 is a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor (TCR). Like the TCR, CD8 binds to a major histocompatibility complex (MHC) molecule but is specific for the class I MHC protein. CD8 is also one of the M1 pro-inflammatory markers. To assess CD8 levels, BV-2 cells were exposed to 100 ng/ml of LPS with or without CB86 and CB204 . The lev3.7Effects of the novel TSPO ligands  and NSAID (DS) on cell metabolism, including mitochondrial metabolism, were compared in LPS-induced BV-2 cells . The cel3.8This experiment was performed to assess the involvement of M2 pathway after following the treatment of 2,4-Di-Cl-MGV-1 along with LPS exposure. Exposure of the BV-2 cells to IL-4 was used as a positive control and LPS as negative control. IL-10  A is one4Our previous study has shown that TSPO ligands 2-Cl-MGV-1 and MGV-1 can counteract with microglial activation and have anti-inflammatory effects . In the In a recent study, we have shown that the TSPO ligands 2-Cl-MGV-1 and MGV-1 counteract with microglial activation including the release of pro-inflammatory cytokines . We relaAttenuation of M1 phenotype while polarizing M2 phenotype is suggested to be a potential approach for the treatment of neuroinflammatory disorders . Just li5In conclusion, our current study shows that the TSPO ligands 2,4-Di-Cl-MGV-1 and CB86 are efficacious in attenuating the activation of microglia and the corresponding release of pro-inflammatory cytokines. This anti-neuroinflammatory activity of the TSPO ligands is reflected by the prevention of the production and release of several inflammatory markers including NO, pro-inflammatory cytokines , NF-Κb p65 (pS536), CD8 and COX-2. Even the 1 μM of 2,4-Di-Cl-MGV-1 significantly inhibited the NO production . This ma10.13039/501100003977Israel Science Foundation .This study was supported by The S.M. designed, performed the experiments and wrote the first draft of the manuscript. This project was supervised by M.G., R.N., and A.W. Methodology and data curation, S.M. and M.G.; writing—review and editing, M.G., A.W., N.D., V.L. and M.F. The TSPO ligands BC86 and CB204 were synthesized by N.D. and V.L. The research was conducted in the laboratory of M.G. The 2,4-Di-Cl-MGV-1 TSPO ligand was synthesized in the Laboratory of I.M by S.S. All authors contributed to the preparation of the final version of the manuscript.The authors declare no conflict of interest.


They show quite different Mo—N and Mo—S distances.Two isomers of the molybdenum(II) complex Mo(CO)(C 2-but-2-yne)carbonylbismolybdenum(II), [Mo(C11H12NOS)2(C4H6)(CO)] or Mo(CO)(C2Me2)(S-Phoz)2, are presented. The N,N-cis–S,S-trans isomer 1 shows quite different bond lengths to the metal atom . In the N,N-trans–S,S-cis isomer 2, which is isotypic with the corresponding W complex, the Mo—N bond lengths [2.236 (2) and 2.203 (2) Å], as well as the Mo—S bond lengths [2.5254 (8) and 2.5297 (8) Å], are almost the same.The synthesis and structural determination of two isomers of the molybdenum(II) complex (η This behaviour is different from that observed for the W variant which crystallized solely as the N,N-trans isomer and showed the presence of a second isomer in solution only to a minor extent.Herein we report an improved synthetic procedure for Mo(CO)via a Pure Solv Solvent Purification System. Chemicals were purchased from commercial sources and used without further purification. The precursor MoI2(CO)3(NCMe)2 was synthesized according to a literature procedure 2, a slight modification of a published procedure was used , doublet (d), doublet of doublets (dd) or multiplet (m). NMR solvents were stored over molecular sieves. Solid-state IR spectra were measured on a Bruker ALPHA ATR–FT–IR spectrometer at a resolution of 2 cm−1. The relative intensity of signals is declared as strong (s), medium (m) and weak (w). Electron impact mass spectroscopy (EI–MS) measurements were performed with an Agilent 5973 MSD mass spectrometer with a push rod.Synthetic manipulations were performed under a nitrogen atmosphere using standard Schlenk and glove-box techniques. Solvents were purified 2(CO)3(NCMe)2  in MeCN (8 ml). The resulting blood-red solution was stirred for 2 h at 35 °C, whereupon the solvent was removed by evaporation. The residue was suspended in toluene (20 ml) and the resulting suspension was filtered through Celite. The blood-red filtrate was then evaporated to dryness. After repeated recrystallization from CH2Cl2/heptane at −25 °C, Mo(CO)2(S-Phoz)2  was obtained as dark red crystals. NMR and IR data are in agreement with previously published results   in MeCN (8 ml) was added dropwise to a solution of MoI2(S-Phoz)2  was dissolved in CH2Cl2 (20 ml), whereupon 2-butyne  was added to the solution at 0 °C under stirring. The cooling bath was removed and the solution was heated under reflux for 24 h. Evaporation of the solvent gave a dark brown powder. Single crystals suitable for X-ray diffraction were obtained from CH2Cl2/heptane solutions at −35 °C. Crystals of both isomers (green plates of 1 and yellow needles of 2) were obtained from the same batch. The product is very sensitive to air and should be stored in a glove-box.Mo(CO)1H NMR for 1 : δ 8.07 , 7.78–7.72 , 7.35 , 7.32–7.27 , 7.21–7.01 , 4.46 , 4.18 , 4.11 , 3.78 , 2.70 , 2.55 , 1.89 , 1.81 , 1.57 , 1.44 ; 1H NMR for 2 : δ 7.67–7.62 , 7.43 , 7.21–7.01 , 6.90–6.84 , 4.11 , 3.93–3.90 , 2.90 , 2.46 , 1.63 , 1.34 , 0.77 , 0.58 . IR (cm−1): 2995 (w), 2962 (w), 2928 (w), 2916 (w), 2894 (w), 1898 , 1856 , 1590 (s), 1572 (s), 1539 , 1455 (m), 1357 (m), 1326 (m), 1280 (m), 1246 (m), 1208 (m), 1160 (m), 1139 (m), 1053 (s), 966 (m), 818 (m), 776 (m), 741 (s), 695 (m), 653 (m). EI–MS (70 eV) m/z: [M – 2CO + O]+ 526.1.2 groups were placed at positions with approximately tetrahedral angles and C—H distances of 0.99 Å, and common isotropic displacement parameters were refined for the H atoms of the same group. The H atoms of the arene rings were placed at the external bisectors of the C—C—C angles at C—H distances of 0.95 Å, and common isotropic displacement parameters were refined for the H atoms of the same ring. The H atoms of the methyl groups were refined with common isotropic displacement parameters for the H atoms of the same group and idealized geometries with tetrahedral angles, enabling rotations around the C—C bonds, and with C—H distances of 0.98 Å.Crystal data, data collection, and structure refinement details are summarized in Table 11 and 2 crystallize without any solvent molecules in the monoclinic space groups P21/n and P21/c, respectively, and both have one metal complex in the asymmetric unit. In N,N-cis isomer 1  Å] is much longer than that trans to the carbonyl ligand [Mo1—N33 = 2.3404 (11) Å]. In N,N-trans isomer 2  Å and Mo1—S2 = 2.3665 (3) Å] than in isomer 2 [Mo1—S1 = 2.5254 (8) Å and Mo1—S2 = 2.5297 (8) Å] or in the W compound [W—S = 2.5232 (4)–2.5243 (4) Å]. On the other hand, in both isomers, the distances are almost the same between the central atom and the butyne ligands [2.0310 (12)–2.0664 (12) versus 2.024 (3)–2.059 (3) Å] and to the carbonyl ligands [1.9417 (13) versus 1.953 (3) Å], although both are arranged in trans positions with respect to the N atoms of the oxazole rings in 1, and trans to the S atoms of the benzenethiolate groups in 2. In both isomers, the CO ligands [C3—O3 = 1.1555 (16) and 1.157 (3) Å] lie roughly in the best planes through the butyne ligands [C1—C2 = 1.2965 (18) and 1.314 (4) Å] and the Mo atoms.Isomers  1 Fig. 1, the Mo— 2 Fig. 2, these dM(CO)(C2R2)(S-Phoz)2 complexes . In isomer 1, the Mo—N distance of the S-Phoz ligand trans to the butyne ligand is exceptionally large due to the wide C1—Mo1—N13 angle of 173.53 (4)° and large C—M—S1 angles. The Mo—S distances are the same in the N,N-trans conformers, but in the S,S-trans conformers, M—S1 is distinctly longer than M—S2. Therefore, the S-Phoz ligands whose oxazole rings are trans to the alkyne ligands are more weakly bound to the metal centre than the others. In all six complexes (C2Me2)(S-Phoz)2, while a 1:1 ratio is observed in CDCl3. The NMR data of isomer 2, which presumably adopts the N,N-trans configuration, are almost identical with those of the W analogue (C2Me2)(S-Phoz)2 exhibit a 95:5 ratio, with a clear preference for the N,N-trans configuration of isomer 2.2Me2)(S-Phoz)2 shows a very strong band at 1898 cm−1 which is attributed to the C≡O bond. Due to weaker π-backbonding of the Mo centre, this bond is stronger by 18 cm−1 compared to that in the respective W compound  1, 2, global. DOI: 10.1107/S2053229622002029/wv30081sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2053229622002029/wv30082sup3.hklStructure factors: contains datablock(s) 2. DOI: 2153636, 2153635CCDC references: