NF2 and PAK1 reciprocally regulate each other in a positive feedback manner. Thus, the aim of the present study is to investigate the effects of PLPP/CIN-mediated NF2 S10 dephosphorylation on PAK1-related signaling pathways under physiological and neuroinflammatory conditions, which are largely unknown.Pyridoxal-5′-phosphate phosphatase/chronophin (PLPP/CIN) selectively dephosphorylates serine (S) 10 site on neurofibromin 2  p65 S276 phosphorylation, cyclooxygenase-2 (COX-2) upregulation, prostaglandin E synthase 2 (PTGES2) induction and neuronal damage were measured. The effects of 1,1'-dithiodi-2-naphthtol  pretreatment on these responses to KA were also validated.After kainate (KA) injection in wild-type, PLPP/CIN overexpression increased PAK1 S204 autophosphorylation concomitant with the enhanced NF2 S10 dephosphorylation in hippocampal neurons under physiological condition. Following KA treatment, PLPP/CIN overexpression delayed the seizure on-set and accelerated PAK1 S204 phosphorylation, NF-κB p65 S276 phosphorylation, COX-2 upregulation and PTGES2 induction, which were ameliorated by PLPP/CIN deletion or IPA-3. Furthermore, IPA-3 pretreatment shortened the latency of seizure on-set without affecting seizure severity (intensity) and ameliorated CA3 neuronal death induced by KA.These findings indicate that PLPP/CIN may regulate seizure susceptibility (the latency of seizure on-set) and CA3 neuronal death in response to KA through NF2-PAK1-NF-κB-COX-2-PTGES2 signaling pathway.The online version contains supplementary material available at 10.1186/s12974-023-02788-9. PLPP/CI6) –6. PLPP/6) . Interes6) , 11. Fur6) –11. Thes6) –14. Sinc6) , PAK1 pl6) , 17. PAK6) . During 6) , 19.moesin-ezrin-radixin-like protein) or schwannomin) is a tumor suppressor protein encoded by the neurofibromatosis type 2 gene NF2. Deletion or loss-of-function mutation of NF2 causes neurofibromatosis type 2, which is a dominant inherited disorder that is characterized by the development of multiple benign tumors of the nervous system. NF2 also affects neuronal excitability by regulating F-actin stability and murine double minute-2 (Mdm2)-mediated postsynaptic density 95 (PSD95) degradation [LIN-11, Isl-1 and MEC-3 domain kinase 1 (LIMK1) that abrogates F-actin depolymerizing activity of cofilin by S3 phosphorylation [Neurofibromin 2  p65 S276 phosphorylation, cyclooxygenase-2 (COX-2) upregulation and prostaglandin E synthase 2 (PTGES2) induction. Furthermore, 1,1'-dithiodi-2-naphthtol  shorPLPP/CIN−/− (129/SvEv-C57BL/6 J background) and PLPP/CINTg (C57BL/6J background) mice (8 weeks old) were used in the present study. Wild-type (WT) mice obtained from 129/SvEv-C57BL/6 J and C57BL/6J strains were separately maintained and used as control animals for PLPP/CIN−/− and PLPP/CINTg mice, respectively. Animals were housed in a standard environment (humidity of 55 ± 5% and a temperature of 22 ± 2 °C on a 12 h light/dark cycle) and provided food and water ad libitum. All animal studies were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of Hallym University . Every effort was made to reduce the number of animals employed and to minimize animal discomfort. All reagents were obtained from Sigma-Aldrich , except as noted.Male 2O:O2). Monopolar electrode  was stereotaxically implanted into the left dorsal hippocampus . The reference electrode was placed in the posterior cranium over the cerebellum. Three days after surgery when baseline electroencephalogram (EEG) recovers from post-surgery peroxisomal discharges [PLPP/CINTg mice were given IPA-3  or v studies –10. EEG  studies . After r studies –10.Animals were decapitated under urethane anesthesia . Thereafter, the hippocampi were rapidly dissected and homogenized in lysis buffer. Lysis buffer contained protease inhibitor cocktail  and phosphatase inhibitor cocktail . Protein concentration was determined using a Micro BCA Protein Assay Kit . An equal amount 10 μg) was loaded on a Bis–Tris sodium dodecyl sulfate-poly-acrylamide gel (SDS-PAGE). The proteins were separated by electrophoresis and transferred to membranes. The membranes were blocked with Tris-buffered saline  containing 2% bovine serum albumin and then incubated with primary antibodies (Table  μg was l2/area) were randomly selected in the CA3 region . Thereafter, mean intensity on each section was measured by using AxioVision Rel. 4.8 and ImageJ software. Intensity measurements were represented as the number of a 256-Gy scale.Animals were injected with KA  or normal saline 20 min after IPA-3  or vehicle treatment. Two hours after KA injection, animals were perfused with 4% paraformaldehyde through the ascending aorta under deep anesthesia with urethane . The brains were then removed and post-fixed in the same fixative for overnight and left in 30% sucrose in phosphate buffer (PB) until sunk. Coronal Sects. (30 μm) were cut using a cryostat. Sections were placed in a plate, rinsed with PBS over 10 min and subsequently blocked for 30 min at room temperature in 10% goat serum . After blocking, sections were incubated with a cocktail solution containing primary antibodies (Table 4 μm2) were captured, and were selected. Thereafter, cells count was performed using AxioVision Rel. 4.8 software [To analyze the KA-induced neuronal damage, we applied Fluoro-Jade B (FJB) staining. Animals used for FJB staining were given diazepam  2 h after KA injection to control seizure activity. Three days after KA treatment, sections were prepared as the same method as aforementioned. Sections were placed on slides, dried, and immersed in 80% ethanol containing 1% sodium hydroxide. Next, slides were immersed in 70% ethanol solution for 2 min and in purified water for 2 min. After immersion in 0.06% potassium permanganate solution for another 10 min, slides were rinsed with purified water for 2 min. Then, slides were incubated for 30 min in 0.001% FJB , freshly prepared by adding 20 ml of a 0.01% stock FJB solution to 180 ml of 0.1% acetic acid, with gentle shaking in the dark. After mounting, areas of interest  followed by Newman-Keuls post-hoc test was used to analyze statistical significance. Linear regression analysis (correlation test) was also performed using data obtained from the same animal. A p < 0.05 was considered to be statistically different [After evaluating the values on normality using Shapiro–Wilk ifferent –10.PLPP/CINTg mice was 0.65-fold of WT mice level under physiological condition (t(12) = 7.07, p < 0.001 vs. WT mice, Student’s t-test, n = 7, respectively; Fig. PLPP/CIN−/− mice was 1.32-fold higher than that in WT mice (t(12) = 9.14, p < 0.001 vs. WT mice, Student’s t-test, n = 7, respectively; Fig. PLPP/CINTg mice was 1.42-fold of WT mice level (t(12) = 9.07, p < 0.001 vs. WT mice, Student’s t-test, n = 7, respectively; Fig. PLPP/CIN−/− mice was 0.66-fold of WT mice level (t(12) = 9.14, p < 0.001 vs. WT mice, Student’s t-test, n = 7, respectively; Fig. R =—0.91, t(26) = 11.46, p < 0.001, n = 7, respectively; Fig. Recently, we have reported that PLPP/CIN dephosphorylates NF2 S10 site without altering its S518 phosphorylation . NF2-medPLPP/CINTg mice showed the longer latency of seizure on-set (t(12) = 8.1, p < 0.001, Student’s t-test, n = 7, respectively; Fig. F = 9.53, p = 0.009, repeated measure one-way ANOVA, n = 7, respectively; Fig. χ2(1) = 10.07, p = 0.018, Friedman test, n = 7, respectively; Fig. PLPP/CIN−/− mice showed the decrease in the latency of seizure on-set (t(12) = 14.22, p < 0.001, Student’s t-test, n = 7, respectively; Fig. F = 11.27, p = 0.006, repeated measure one-way ANOVA, n = 7, respectively; Fig. χ2(1) = 12.45, p = 0.006, Friedman test, n = 7, respectively; Fig. To further explore the role of PLPP/CIN-mediated NF2 S10 dephosphorylation in PAK1 activity, we investigated whether the altered NF2 S10 phosphorylation is relevant to the changed PAK1 S204 phosphorylation following KA injection. Consistent with our previous studies –10, PLPPPLPP/CINTg mice, respectively  = 386.08, p < 0.001; FKA = 27.41, p < 0.001; Fgroup*KA = 20.67, p < 0.001; two-way ANOVA, n = 7, respectively; Fig. PLPP/CINTg mice, respectively  = 216.81, p < 0.001; FKA = 179.74, p < 0.001; Fgroup*KA = 5.43, p = 0.029; two-way ANOVA, n = 7, respectively; Fig. PLPP/CINTg mice, respectively  = 71.35, p < 0.001; FKA = 7.69, p < 0.001; Fgroup*KA = 90.04, p = 0.011; two-way ANOVA, n = 7, respectively; Fig. R = − 0.88, t(12) = 6.408, p < 0.001, n = 7, respectively; Fig. PLPP/CINTg mice  = 4.19, p = 0.001, n = 7, respectively; Fig. Compatible with our previous studies , 11, KA  = 1315.08, p < 0.001; FKA = 20.49, p < 0.001; Fgroup*KA = 158.23, p < 0.001; two-way ANOVA, n = 7, respectively; Fig. PLPP/CIN−/− mice  = 971.14, p < 0.001; FKA = 128.85, p < 0.001; Fgroup*KA = 109.03, p < 0.001; two-way ANOVA, n = 7, respectively; Fig. In PLPP/CINTg mice 20 min prior to KA injection. Although IPA-3 pretreatment did not affect the basal EEG level, it decreased the latency of seizure on-set in response to KA from 14.5 to 8.8 min in WT mice and from 27.8 to 17.7 min in PLPP/CINTg mice, respectively  = 124.76, p < 0.001; Ftreatment = 63.39, p < 0.001; Fgroup*treatment = 4.94, p = 0.036; two-way ANOVA, n = 7, respectively; Fig. PLPP/CINTg mice , which is permeable to the brain-blood barrier –31, in WNF-κB is involved in the regulation of seizure activity and postictal events. Briefly, KA acutely activates NF-κB in hippocampal neurons , and NF-PLPP/CINTg mice was 1.36-fold of WT mice level. KA increased PAK1 S204 phosphorylation level to 1.38- and 1.6-fold of control WT mice level in WT and PLPP/CINTg mice, respectively  = 172.73, p < 0.001; FKA = 198.81, p < 0.001; Fgroup*KA = 10.24, p = 0.004; two-way ANOVA, n = 7, respectively; Fig. PLPP/CINTg mice following KA treatment, respectively  = 231.56, p < 0.001; Ftreatment = 104.46, p < 0.001; Fgroup*treatment = 7.02, p = 0.003; two-way ANOVA, n = 7, respectively; Fig. PLPP/CINTg mice was 1.46-fold of WT mice level under physiological condition. KA increased p65 S276 phosphorylation level to 1.44- and 1.65-fold of control WT mice level in WT and PLPP/CINTg mice, respectively  = 117, p < 0.001; FKA = 104.11, p < 0.001; Fgroup*KA = 15.5, p < 0.001; two-way ANOVA, n = 7, respectively; Fig. PLPP/CINTg mice following KA treatment, respectively  = 193.59, p < 0.001; Ftreatment = 57.29, p < 0.001; Fgroup*treatment = 8.52, p < 0.001; two-way ANOVA, n = 7, respectively; Fig. Under physiological condition, PAK1 S204 phosphorylation level in CA3 neurons of PLPP/CINTg mice  = 88.16, p < 0.001; Ftreatment = 65.51, p < 0.001; Fgroup*treatment = 1.78, p = 0.184; two-way ANOVA, n = 7, respectively; Fig. PLPP/CINTg mice following KA injection  = 153.12, p < 0.001; Ftreatment = 70.1, p < 0.001; Fgroup*treatment = 0.38, p = 0.686; two-way ANOVA, n = 7, respectively; Fig. Western blot also revealed that KA increased PAK1 S204 phosphorylation level following KA treatment, which was attenuated IPA-3 in both WT and R = 0.868, t(19) = 7.619, p < 0.001, n = 7, respectively; Fig. PLPP/CINTg mice  = 3.714, p = 0.001, n = 7, respectively; Fig. R = 0.869, t(26) = 8.96, p < 0.001, n = 7, respectively; Fig. R = 0.772, t(26) = 6.19, p < 0.001, n = 7, respectively; Fig. Linear regression analysis revealed direct proportions between PAK1 S204 and p65 S276 phosphorylations in WT (2) synthesis, which is catalyzed by PTGES2 in neurons under pathophysiological condition [COX-2−/− mice and COX-2 inhibitors exacerbate seizure activity induced by KA due to the reduced γ-aminobutyric acid type A (GABAA) receptor-mediated inhibitions [COX-2 is the inducible cyclooxygenase isoform that plays a central role in the inflammatory cascade . COX-2 iondition –46. Inteibitions –53. Sincibitions , 55, it PLPP/CINTg mice  = 32.05, p < 0.001; FKA = 210.02, p < 0.001; Fgroup*KA = 31.64, p < 0.001; two-way ANOVA, n = 7, respectively; Fig. PLPP/CINTg mice following KA treatment, respectively  = 55.8, p < 0.001; Ftreatment = 145.76, p < 0.001; Fgroup*treatment = 22.26, p < 0.001; two-way ANOVA, n = 7, respectively; Fig. PLPP/CINTg mice, respectively  = 49.5, p < 0.001; FKA = 234.74, p < 0.001; Fgroup*KA = 51.27, p < 0.001; two-way ANOVA, n = 7, respectively; Fig. PLPP/CINTg mice following KA treatment, respectively  = 82.56, p < 0.001; Ftreatment = 162.64, p < 0.001; Fgroup*treatment = 36.6, p < 0.001; two-way ANOVA, n = 7, respectively; Fig. R = 0.917, t(19) = 10.041, p < 0.001, n = 7, respectively; Fig. PLPP/CINTg mice  = 18.778, p < 0.001, n = 7, respectively; Fig. R = 0.859, t(26) = 8.56, p < 0.001, n = 7, respectively; Fig. R = 0.848, t(26) = 8.16, p < 0.001, n = 7, respectively; Fig. Under physiological condition, there was no difference in COX-2 expression between WT and ice Fig. a, b. KA Fgroup = 73.63, p < 0.001; Ftreatment = 216.43, p < 0.001; Fgroup*treatment = 26.84, p < 0.001; two-way ANOVA, n = 7, respectively; Fig. Fgroup = 112.14, p < 0.001; Ftreatment = 338.92, p < 0.001; Fgroup*treatment = 53.75, p < 0.001; two-way ANOVA, n = 7, respectively; Fig. PLPP/CINTg mice than WT mice 3 days after KA injection, which were attenuated by IPA-3 pretreatment  = 67.18, p < 0.001; Ftreatment = 50.75, p < 0.001; Fgroup*treatment = 0.59, p = 0.451; two-way ANOVA, n = 7, respectively; Fig. PLPP/CIN−/− mice  and seizure-induced CA3 neuronal degeneration in response to KA through NF2-PAK1-NF-κB-COX-2-PTGES2 signaling pathway Fig. .Fig. 8ScAlthough the shorter latency of seizure on-set is generally thought to represent the higher seizure intensity and to exert its progression, many literatures describe the inconsistency between seizure threshold and intensity. Deletion of dopamine β-hydroxylase or norepinephrine transporter deletion decreases seizure threshold and its seizure duration , 57. In Fmr1) KO mice (a Fragile X syndrome model), since FRAX486 (a PAKs inhibitor) reduces audiogenic seizure activity in this model [Fmr1 KO mice in response to KA is similar to that of WT mice [Fmr1 KO mice, the present study reveals that PLPP/CIN-mediated PAK1 S204 phosphorylation may increase the latency of seizure on-set, and that IPA-3 increased seizure susceptibility in response to KA without seizure intensity and its progression, suggesting that PLPP/CIN-NF2-PAK1 signaling pathway may modulate seizure susceptibility in response to KA.Recently, we have reported that PLPP/CIN selectively dephosphorylates NF2 S10 site, which increases seizure intensity and its progression in response to KA by regulating F-actin stability, NMDAR-PSD95 co-assembly and NF2-mediated Mdm2 degradation . In the is model , 64. How WT mice . Unlike 2 possesses anti-convulsive properties in acute seizure models [2 also mediates neuroinflammation leading to neuronal damage [PLPP/CINTg mice, while it shortened the latency of KA-induced seizure on-set without altering seizure intensity and behavioral seizure severity in both strains. This inconsistency between the latency of seizure on-set and CA3 neuronal damage indicates that protective effect of IPA-3 on seizure-induced CA3 neuronal degeneration may be relevant to COX-2-PTGES2-mediated events rather than the degree of seizure activity/duration. Taken together, our findings suggest that PLPP/CIN-mediated regulation of NF2-PAK1-NF-κB-COX-2-PTGES2 may decrease seizure susceptibility to KA, but enhance the subsequent inflammatory responses leading to seizure-induced CA3 neuronal death.It has been well known that NF2 inhibits PAK1-mediated NF-κB transactivation , 37, 40.e models , 49, 66.l damage . KA immel damage , 67–69. PLPP/CIN−/− mice, although it increased NF2 S10 phosphorylation level. Considering weak seizure activity in PLPP/CIN−/− mice in response to KA, it is plausible that the low seizure intensity may lead to the dissociation between NF2 dephosphorylation and PAK1 phosphorylation. Adversely, the increased NF2 S10 phosphorylation induced by KA may be insufficient to induce PAK1 S204 phosphorylation or be involved in F-actin stability rather than PAK1 autophosphorylation [PLPP/CIN−/− mice. However, it is not excluded the possibility that other signaling molecules would be involved in the regulation of KA-induced PAK1 autophosphorylation, independent of NF2. Further studies are needed to elucidate NF2-independent regulation of PAK1 autophosphorylation in PLPP/CIN−/− mice.On the other hand, KA did not affect PAK1 S204 phosphorylation in rylation  in PLPP/The present data we demonstrate for the first time that PLPP/CIN-mediated NF2 S10 dephosphorylation may reduce seizure susceptibility and augment CA3 neuronal damage in response to KA by activating PAK1-NF-κB-COX-2-PTGES signaling pathway. Therefore, detailed elucidation of this pathway will be of great importance in understanding the underlying mechanisms of ictogenesis and seizure-induced neuronal death.Additional file 1: Figure S1. Full-length gel images of Western blots in Fig. 1. Figure S2. Full-length gel images of Western blots in Fig. 2. Figure S3. Full-length gel images of Western blots in Fig. 3. Figure S4. Full-length gel images of Western blots in Fig. 5. Figure S5. Full-length gel images of Western blots in Fig. 7.


Commercial severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody tests were developed before variants with spike protein mutations emerged, leading to concerns that these tests have reduced sensitivity for detecting antibody responses in individuals infected with Omicron subvariants. This study was performed to evaluate Abbott ARCHITECT serologic assays, AdviseDx SARS-CoV-2 IgG II, and SARS-CoV-2 IgG for the detection of spike (S) and nucleocapsid (N) IgG antibody increases in vaccinated healthcare workers infected with Omicron subvariants.During the BA.1/2 and BA.4/5 waves, 171 SARS-CoV-2-infected individuals  were tested for S and N IgG post infection. Sequencing and SARS-CoV-2 variant confirmation were performed on nasal swab samples from individuals infected during the BA.1/2 wave.P < 0.001) during the BA.1/2 wave, and 3.6-fold from 1771 ± 351 BAU/ml to 8224 ± 943 BAU/ml (P < 0.001) during the BA.4/5 wave. N IgG increased post infection 19.1-fold from 0.2 ± 0.1 to 3.7 ± 0.5 (P < 0.001) during the BA.1/2 wave and 13.5-fold from 0.22 ± 0.1 to 3.2 ± 0.3 (P < 0.001) during the BA.4/5 wave. Among 159 infection-naïve individuals, positive N IgG levels were detected with a sensitivity of 88% in the 87 individuals who were tested between 14 days and 60 days post infection.Twenty-seven Omicron sequence confirmed individuals in the BA.1/2 wave and all 49 in the BA.4/5 wave had pre-infection antibody data. Compared to pre-infection levels, post-infection S IgG increased 6.6-fold from 1294 ± 302 BAU/ml (mean ± standard error measurement) to 9796 ± 1252 BAU/ml (The large increases in post-infection S IgG along with the N IgG sensitivity that was comparable to previously reported N IgG sensitivity data in unvaccinated individuals after Omicron infection, support the use of Abbott SARS-CoV-2 assays for detecting increased S IgG and seroconversion of N IgG in vaccinated individuals post Omicron infection. Given that 68% of the United States population is fully vaccinated, these results are of current relevance. During the Omicron BA.1/2 wave in the Chicago, Illinois metropolitan area (December 2021 to May 2022) For comparison between the previously infected and infection-naïve groups, participants were considered previously infected if they had a history of positive SARS-CoV-2 PCR, positive antigen testing, or positive N IgG results prior to their presumed Omicron infection. Participants were considered infection-naïve if they had a negative history SAR-CoV-2 PCR or antigen testing, and negative N IgG results prior to their presumed Omicron infection.S and N IgG were measured with the AdviseDx SARS-CoV-2 IgG II (S) and SARS-CoV-2 IgG (N) assays on an Abbott ARCHITECT i2000SR, as described previously. t-test, Chi-square test, and Mann–Whitney U-test. Significance was defined as P < 0.05.The statistical analysis was performed using GraphPad Prism, with the Student paired 3All 171 participants had received at least two doses of an mRNA vaccine prior to enrollment into this study. P > 0.05, for S IgG and for N IgG). For the one sequenced Delta variant, S IgG was 6522 BAU/ml and N IgG was 5.7.P < 0.001) than pre-infection levels (1294 ± 302 BAU/ml) (P < 0.001) than pre-infection levels (0.2 ± 0.1)  A, and po2 ± 0.1) B.Figure n = 17) or rapid antigen testing (n = 32). All 49 individuals had pre-infection antibody data . Post infection, S IgG increased 3.6-fold from 1771 ± 351 BAU/ml to 8224 ± 943 BAU/ml (P < 0.001) (P < 0.001) (P = 0.01), but the post-infection N IgG levels did not differ significantly between the two waves (P > 0.05).During the BA.4/5 wave, 49 individuals were identified in the longitudinal vaccine antibody study who tested positive for SARS-CoV-2 by PCR (< 0.001) C and N I< 0.001) D. Post-ir = 0.38, P = 0.053 and r = 0.08, P = 0.57, respectively).Spearman's correlation test was performed to determine whether post-infection S IgG levels correlated with post-infection N IgG levels for both Omicron waves. There was no correlation between S IgG levels and N IgG levels post infection for the BA.1/2 wave or for the BA.4/5 wave . During the BA.1/2 wave, post-infection S IgG levels did not differ significantly between previously infected (11 715 ± 8075 BAU/ml) and naïve (11 645 ± 877 BAU/ml) participants (P = 0.99). The difference in N IgG levels between previously infected (4.9 ± 1.8) and infection-naïve (3.1 ± 0.2) participants was also not significant (P = 0.053). During the BA.4/5 wave, post-infection S IgG levels did not differ significantly between previously infected (3816 ± 1471 BAU/ml) and naïve (8840 ± 1024 BAU/ml) participants (P = 0.08) . However, N IgG levels were significantly higher in previously infected participants (5.0 ± 1.1) compared to infection-naïve participants (2.9 ± 0.3) (P = 0.02) . These statistical comparisons might not be representative of a larger population given the small sample size of the previously infected populations (six for each wave).It was next investigated whether participants who had SARS-CoV-2 infections prior to their Omicron infection responded differently in terms of S IgG compared to infection-naïve participants. Of the 99 participants (from both Omicron waves) who had pre- and post-Omicron infection antibody data, 12 (six in each wave) had a SARS-CoV-2 infection prior to enrolling in this study. Ten of 12 (83.3%) previously infected individuals compared to 85 of 87 (97.7%) naïve individuals had increased S IgG post Omicron infection  had post-infection positive N IgG. The sensitivity increased to 88.5% (77/87) when tested 14–60 days post infection. Among the 159 infection-naïve individuals, sensitivity was 77.4%. The sensitivity increased to 88% (66/75) when tested 14–60 days post infection. Among the 12 previously infected individuals, four had pre-infection positive N IgG and three of these four (75%) had an increase of ≥1.4 (S/C index) post infection. Eight had pre-infection N IgG <1.4 index and seven of these eight (87.5%) seroconverted. A subset of 35 participants during the BA.1/2 wave and 38 participants during the BA.4/5 wave had their post-infection plasma tested with the Panbio lateral flow anti-S antibody assay. All 73 samples were positive on the Panbio assay. The S IgG for these participants ranged from 613 BAU/ml to 54 175 BAU/ml.4We previously reported that vaccinated individuals infected during the Omicron BA.1/2 wave had significantly higher S IgG (8304 BAU/ml) than individuals infected prior to the Omicron BA.1/2 wave (3740 BAU/ml) Post-Omicron infection positive N IgG was detected with a sensitivity of 77.4% (88% when tested 14–60 days post infection) among the infection-naïve vaccinated individuals. These findings are similar to a previously reported sensitivity of 84% in 37 infection-naïve unvaccinated individuals Study limitations include a limited study size with a single cohort of participants. This cohort was of low demographic diversity and thus the results cannot be generalized to male individuals and non-White populations. Viral samples during the Omicron BA.4/5 wave were not available for sequencing; however, during this time >75% of Chicago area This work demonstrates that previous observations of reduced antibody detection among vaccine/infection-naïve individuals who were infected with Omicron The authors declare the following financial interests/personal relationships: MA, MR, AO, BH, MS, VH, AA, EG, TF, EA, and GC are employees and shareholders of Abbott Laboratories. This work was funded by Abbott Laboratories. AL is a consultant for Abbott Laboratories.


To determine the mechanisms underlying these promising effects, we investigated metabolic characteristics. Cytokine secretion by γδ-TCR-stimulated nongene-modified γδ-T cells (NGMCs) and αβ-TCR-stimulated GMCs was equally suppressed by a glycolysis inhibitor, although the cytokine secretion of αβ-TCR-stimulated GMCs was more strongly inhibited by ATP synthase inhibitors than that of γδ-TCR-stimulated NGMCs. Metabolomic and transcriptomic analyses, flow cytometry analysis using mitochondria-labeling dyes and extracellular flux analysis consistently suggest that αβ-TCR-transduced γδ-T cells acquire superior mitochondrial function.Adoptive cell therapy using allogeneic γδ-T cells is a promising option for off-the-shelf T cell products with a low risk of graft-versus-host disease (GVHD). Long-term persistence may boost the clinical development of γδ-T cell products. In this study, we found that genetically modified Vγ9In conclusion, αβ-TCR-transduced γδ-T cells acquire superior mitochondrial function with promising persistence.  •Long-term persistence may boost the clinical development of γδ-T cell products•αβ-TCR transduction into γδ-T cells showed promising persistence•αβ-TCR transduced γδ-T cells could utilize more mitochondrial energy metabolism Cellular therapy; Molecular biology; Immunology This TCR had two amino acid replacements in the CDR2 region of the TCRβ chain to produce high affinity,,,,A new era of immunotherapy for cancer has arrived with the application of immune checkpoint inhibitors and adoptive cell therapy with genetically modified T cells directed against cancer antigens. Chimeric antigen receptor-engineered T cells (CAR-T cells) targeting CD19 have shown clinical benefits in clinical trials,,,,,,,Despite the successes described previously, there are two main issues with adoptive T cell therapy using αβ-T cells: mispairing of endogenous and engineered αβ-TCRs+Vδ2+ T cells expressing NY-ESO-1-specific αβ-TCR and the CD8 coreceptor (NE1-GMCs), with a specific focus on their metabolic preferences in association with their effector function and persistence.In this study, we evaluated genetically modified Vγ9+Vδ2+ phenotype (data not shown). Since coexpression of CD8αβ enhanced the antitumor effect of NY-ESO-1-specific αβ-TCR (G50A + A51E TCR)-transduced γδ-T cells  ethylidene-1,1-bisphosphonate (PTA),-T cells A–S1D, we-T cells A. In NE1gh cells B. Transdgh cells C and 1D,gh cells A. Transdgh cells B–S2D. NEgh cells C. NE1-GMor cells A–S3C. Toadiation E. NE1-GMadiation F and S3DNE1-pMHC G.Figure In cultures without cytokine supplementation, cell growth of PTA-treated γδ-T cells was suppressed A and S4BTo assess the dependence on glucose metabolism, examinations of cytokine secretion by γδ-T cells using metabolic inhibitors were performed. IFN-γ secretion by NGMCs and NE1-GMCs decreased with increasing concentrations of 2-deoxy-D-glucose (2-DG), a glycolysis inhibitor A. Compar+ cells in NGMCs and CD8+ cells in NE1-GMCs used in the study were similar , and 357 metabolites  were detected A. Metabo similar A and S6BRNA microarray analysis was performed using cells from the four groups as in the metabolomic analysis. Cells were newly induced on different days from those used for metabolomic analysis. Gene set enrichment analysis (GSEA) showed a trend toward increased expression of genes related to mitochondria in NE1-GMCs compared to NGMCs. Gene expression associated with the “electron transport chain: oxphos system in mitochondria” of WikiPathways was higher in NE1-GMCs than in NGMCs under both unstimulated and stimulated conditions . Gene exThe mitochondrial phenotype of NGMCs and NE1-GMCs was assessed using MitoTracker Green and tetramethylrhodamine methyl ester (TMEM). NE1-GMCs had higher mitochondrial mass and mitochondrial membrane potential in the unstimulated state A. AnotheCellular metabolic status was assessed by extracellular flux analysis, measuring the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), with the results representing the levels of mitochondrial respiration and glycolysis, respectively. In the absence of TCR stimulation, the median basal OCR/basal ECAR ratio of NGMCs was similar to or higher than that of NE1-GMCs (NGMCs: 0.95 vs. NE1-GMCs: 0.65) B and S7Bin vivo persistence of αβ-T cells is thought to be related to antitumor efficacy, and mainly in the context of CAR-T cell development, incorporation of 4-1BB (CD137) signaling,,+Vδ2+ T cells are an innate-like T cell population.in vitro and in vivo. Lope et al. reported that IFN-γ secretion by γδ-T cells was glycolysis-dependent,,In a study on TCR-T cells, an autoreactive TCR consisting of endogenous TCR and engineered TCR caused lethal toxicity in a mouse model.In our study, αβ-TCR transduction increased the mitochondrial mass and mitochondrial membrane potential of γδ-T cells. αβ-TCR-transduced γδ-T cells contained more metabolites in the TCA cycle and OXPHOS and had higher gene expression of the mitochondrial electron transport chain in both unstimulated and stimulated states. In the stimulated state, IFN-γ production was more dependent on ATP synthesis, which was supported by the higher OXPHOS gene expression observed in αβ-TCR-transduced γδ-T cells. Although not high on the list in our gene analysis, our examinations using metabolic inhibitors suggest that energy through glutamine and fatty acid metabolism might also be important for the characteristics of αβ-TCR-transduced γδ-T cells. The metabolism of glutamine and fatty acids in αβ-TCR-transduced γδ-T cells requires further study.,Evaluation of spare respiratory capacity (SRC) is a useful method for assessing mitochondrial function in T cells.in vivo persistence of αβ-T cells.,,,+ T cell effector function and proliferation.Recently, Stenger et al. reported the importance of the presence of αβ-TCR for αβ-T cell persistence by showing that knock out of endogenous TCR in CD19 CAR-T cells resulted in a lack of persistence, although the mechanism remains unclear.in vitro and in vivo persistence. As a mechanism, γδ-T cells could utilize more mitochondrial energy metabolism due to the acquisition of αβ-TCR and CD8.In conclusion, αβ-TCR transduction into γδ-T cells showed promising The study has several limitations: small sample size and reporting from a single group.mishihara@med.mie-u.ac.jp).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Mikiya Ishihara (This study did not generate new unique reagents.scid/IL-2Rγnull (NOG) female mice were purchased from the Central Institute for Experimental Animals . Mice were fed a standard diet, housed under specific pathogen-free conditions, and used at 6–8 weeks of age. Five days after subcutaneous NW-MEL-38 melanoma cell inoculation, the mice were treated with total body irradiation (TBI) (2 Gy) and then infused with or without NE1-GMCs. The number of infused NE1-GMCs was 5×106 cells/mouse. All animal experiments were conducted according to protocols approved by the Animal Care and Use Committee of the Mie University Life Science Center.NOD/Shi-+, TAP-deficient, Epstein–Barr virus-transformed lymphoblastoid cell line), SW982 cells , Fuji cells , and NW-MEL-38 cells  were cultured in RPMI-1640 medium supplemented with 25 mM HEPES, 10% fetal calf serum (FCS), 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. Mycoplasma contamination of the cell lines was checked, but not detected.T2 cells (HLA-A2p157/HLA-A∗02:01-specific TCR-α and TCR-β chains with G50A + A51E amino acid replacements) and human CD8 , TCR αβ-negative cells were cultured in modified Yssel’s medium with 10% human type AB serum and PTA (day 0).uman CD8 A were usAll procedures performed in this study involving human participants were conducted in accordance with the Japanese law of Ethical Guidelines for Medical and Biological Research Involving Human Subjects.p157/HLA-A∗02:01 complex (NE1-pMHC) (1.0 ng/μL)-coated plate. For the intracellular staining of cytokines, γδ-T cells were cultured for 5–6 h with a GolgiStop Protein Transport Inhibitor . For metabolic inhibition experiments, RPMI 1640 (glucose-free) medium, 2-DG , etomoxir, CB-839 , and oligomycin  were used. For mitochondrial phenotype assessment, MitoTracker Green (Invitrogen) and tetramethylrhodamine (TMRM) (FUJIFILM) were used.Cytokine secretion and TCR expression in γδ-T cells were measured using flow cytometry. γδ-T cells were cultured with Fc Receptor Blocking Solution Human TruStain FcX (BioLegend) prior to antibody staining. Fluorescently labeled anti-human TCR Vδ2 monoclonal antibody (mAb) (Clone B6), anti-human CD8a mAb (clone RPA-T8), anti-human CD3ε mAb (clone SK7), anti-human CD45 mAb (clone HI30), anti-human IFN-γ mAb (clone 4S. B3) were purchased from BioLegend, Inc., and anti-Ki67 mAb was purchased from Beckman Coulter, Inc. . The live or dead fixable dead cell staining kit NIR fluorescence  was used to identify the live cells. For T cell stimulation, γδ-T cells were cultured in PTA (1.0 μM)-containing medium on a TCRγδ-specific agonistic mAb (clone IMMU510) (Beckman Coulter) (1.0 μg/mL)-coated plate or NE16 cells per sample, with three samples from three different healthy donors included in each group, and methanol containing an internal standard solution after washing with mannitol-containing solution on day 8. Prior to extraction, T cells were cultured for 6 h in RPMI 1640-10% FCS medium with or without TCR stimulation. TCRγδ-specific agonistic mAb- and NE1-pMHC-coated plates were used for NGMCs and NE1-GMCs, respectively.Comprehensive analysis of hydrophilic and ionic metabolites using a combination of capillary electrophoresis and Fourier transform mass spectrometry (CE-FTMS) was performed by Human Metabolome Technologies, Inc. . NE1-GMCs were purified using CD8 Microbeads (Miltenyi Biotec) on day seven. Metabolic extracts were prepared from 2.0-3.5 × 105 cells per sample from a healthy donor using a QIAamp RNA Blood Mini kit . Prior to RNA extraction, NGMCs and NE1-GMCs purified with CD8 microbeads were cultured with or without TCR stimulation, as in metabolomic analysis. RNA microarray analysis was performed by Agilent Technologies, Inc. Briefly, the RNA integrity of the samples was determined using an Agilent 2100 Bioanalyzer and Agilent RNA 6000 Pico Kit. All samples showed an RNA integrity number (RIN) higher than eight and qualified for further processing. Total RNA was reverse-transcribed to cDNA and then synthesized into Cy3-labeled cRNA by in vitro transcription. Labeled samples were hybridized to a SurePrint G3 Human GE Microarray 8 × 60 K Ver3.0. Scanning was performed using a SureScan Microarray scanner. Gene set enrichment analysis (GSEA)https://www.gsea-msigdb.org/gsea/index.jsp). The log2-transformed, normalized and validated gene data by Agilent Technologies, Inc. was used. The genes were ranked based on the gene set ‘WikiPathways subset of Canonical Pathways’.RNA extracts were prepared from 3.1 × 105 cells/well, with the same number of cells included in each experiment, in a cell culture plate with assay medium and cultured for a total of 50–60 min at 37°C in a CO2-free incubator until analysis. The final concentrations of oligomycin, FCCP, and rotenone/antimycin A were 1.5, 1.0 μM and 0.5 μM, respectively. The basal OCR/basal ECAR ratio was calculated using the equation: (pmol/min)/(mpH/min).OCR and ECAR were measured with a Seahorse XF HS Mini Analyzer using a Seahorse XF Cell Mito Stress Test Kit . Briefly, after thawing, γδ-T cells were cultured in RPMI 1640-10% FCS medium. The TCR stimulation time was 72 h. After washing with assay medium, γδ-T cells were plated at 2.0–3.0 × 10For cytokine inhibition studies, a t test was used if Levene’s test showed a value above the significance level; otherwise, Welch’s t test was used. For metabolic status analysis using Seahorse, the Mann–Whitney U test was used. Calculations were performed using SPSS Statistics version 26 . For the metabolomic analysis, we described the results of Welch’s test reported by Human Metabolome Technologies, Inc. p values less than 0.05 were considered statistically significant .


Chimaera phantasma (silver chimera)  collected off Koshimoda in Suruga Bay in April to May 2022 were comprehensively analyzed. Bacteria belonging to the phylum Proteobacteria were the dominant species. Occupancy rates of other bacterial phyla differed greatly among the samples.The intestinal microbiota of  Chimaera phantasma (silver chimera) (ACACTCTTTCCCTACACGACGCTCTTCCGATCT-NNNNN-CCTACGGGNGGCWGCAG-3′) and 805r (5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-NNNNN-GACTACHVGGGTATCTAATCC-3′) for amplification of the 16S rRNA gene (V3 to V4 regions), 2 μL each of 2 mM deoxynucleoside triphosphates (dNTPs), and 1 μL of the gut content DNA. The reaction conditions were as follows: 94°C for 2 min, 30 cycles of 98°C for 10 s, 55°C for 30 s, and 68°C for 30 s, and finally 68°C for 7 min. The PCR products were purified using the AMPure XP system (Beckman Coulter). The 10-μL second PCR mixture contained 1 μL of 10× Ex Taq buffer, 0.1 μL of Ex Taq high-sensitivity (HS) DNA polymerase (TaKaRa Bio), 1 μL each of primers 2ndF (5′-AATGATACGGCGACCACCGAGATCTACAC-Index2-ACACTCTTTCCCTACACGACGC-3′) and 2ndR (5′-CAAGCAGAAGACGGCATACGAGAT-Index1-GTGACTGGAGTTCAGACGTGTG-3′) (Illumina), 2 μL each of 2.5 mM dNTPs, and 2 μL of the first-round PCR product. The indexed PCR conditions were as follows: 94°C for 2 min, 12 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, and finally 72°C for 5 min. These PCR products were purified using the AMPure XP system and quantified using a QuantiFluor double-stranded DNA (dsDNA) system (Promega) and Synergy H1 plate reader (BioTek). A library quality check was performed using the dsDNA 915 reagent kit and a Fragment Analyzer . The PCR products were sequenced (2 × 300-bp paired-end reads) using the MiSeq reagent kit v.3 and a MiSeq sequencer (Illumina). The reads obtained were demultiplexed and trimmed using fastx_barcode_splitter and fastx_trimmer, respectively, in FASTX-Toolkit v.0.0.14 (https://qiime2.org). Sequences showing ≥99% similarity were grouped into operational taxonomic units (OTUs) using the SILVA database v.138   were cause v.138   of the species in sample C_1 and 26.1% (22 species) of those in sample C_3. Other taxa with occupancy rates of ≥10% were Actinobacteria (19.5%), Firmicutes (11.7%), and Patescibacteria (10.5%) in sample C_1 and Desulfobacterota (35.9%) and Chloroflexi (15.9%) in sample C_3.The three samples contained a total of 123 OTUs. mple C_2 , comprisn oceans –15. ProtDRR413612, DRR413613, and DRR413614.The 16S rRNA gene amplicon sequence data have been deposited in the DDBJ Sequence Read Archive (DRA) under the accession numbers


Variability of gene expression due to stochasticity of transcription or variation of extrinsic signals, termed biological noise, is a potential driving force of cellular differentiation. Utilizing single-cell RNA-sequencing, we develop VarID2 for the quantification of biological noise at single-cell resolution. VarID2 reveals enhanced nuclear versus cytoplasmic noise, and distinct regulatory modes stratified by correlation between noise, expression, and chromatin accessibility. Noise levels are minimal in murine hematopoietic stem cells (HSCs) and increase during differentiation and ageing. Differential noise identifies myeloid-biased Dlk1+ long-term HSCs in aged mice with enhanced quiescence and self-renewal capacity. VarID2 reveals noise dynamics invisible to conventional single-cell transcriptome analysis.The online version contains supplementary material available at 10.1186/s13059-023-02974-1. Single-cell genomics has become a powerful method of choice for the identification of cell types and for the inference of tissue composition at single-cell resolution –3. The rE. coli lac operon  is added to each cell beforehand. Moreover, to account for reusing information across connected cells, the j, a term accounting for the variability in sequencing efficiency or RNA content across individual cells. We generated a simulated data set with 34,390 genes and 100 cells, corresponding to a homogenous neighborhood. Random transcript counts were sampled from a negative binomial distribution with mean  dataset . The meaWe estimated the total dispersion parameter per gene from the reference dataset as mentclass2pt{minimN = 20,000, thinning period length Thin = 20; and length of burn-in period Burn = 10,000.We applied BASiCS , 20 to thttps://www.ncbi.nlm.nih.gov/geo/) or from the 10 × Genomics website (https://support.10xgenomics.com/single-cell-multiome-atac-gex/datasets). See details in Additional file k-nearest neighbor (knn) network of cells was computed with the pruneKnn function, with number of neighbors set to 25. Clustering was performed with the Leiden algorithm for community detection  all PE-Cy7; anti-CD117/c-Kit (2B8) in BV711; anti-Ly6a/Sca-1 (D7)-APCCy7; anti-CD34 (RAM34) in AF700; anti-CD150 (TC15-12F12.2) in PE/Dazzle; anti-CD48 (HM48-1) in BV421. Monoclonal antibodies were purchased from eBioscience, BioLegend, or MBL. Either DLK1 + or DLK1 − HSCs were sorted. Cells were sorted into Stem Pro®-34 SFM (Life Technologies) for further experiments.Murine bone marrow (BM) cells were isolated from pooled femura, tibiae, hips, ilia, and vertebrae by gentle crushing in PBS using a mortar and pistil. Erythrocyte lysis was performed using ACK Lysing Buffer. To enrich for lineage-negative (Lin−Kit+Sca1+CD150+CD48−CD34−) were FACS sorted into 72-well Terasaki plates and cultured in StemPro-34 SFM containing 50 ng/ml SCF, 25 ng/ml TPO, 30 ng/ml Flt3-Ligand, 100 ml/ml Penicillin/Streptomycin, and 2 mM L-Glutamine. After 48 h, each well was checked manually for the number of cell divisions under the microscope: 1 cell = no division, 2 cells = 1 division, > 2 cells =  > 1 division.Single DLK1+ or DLK1− HSCs (Lineage−Kit+Sca1+CD150+CD48−CD34−) were FACS sorted into MethoCult M3434, plated and cultured. Approximately 7 days after the first plating, number of colonies were counted and 10,000 cells were re-plated. Second and third platings were performed 3 and 5 days, respectively, after the first re-plating. Colonies were also quantified at these time points.Two hundred to four hundred DLK1+ and DLK1− HSCs (Lineage−Kit+Sca1+CD150+CD48−CD34−) isolated from 13–15-month-old CD45.2 C57BL/6 mice were transplanted into lethally irradiated (4.5 Gray + 5 Gray) CD45.1 (Ly5.1) mice together with 5 × 105 supportive spleen cells from 8–12-week-old CD45.1/2 mice within 24 h after irradiation by intravenous tail vein injection. Contribution of donor cells (CD45.2) was monitored in peripheral blood at 4, 8, 12, and 16 weeks post transplantation. For endpoint analysis, bone marrow was analyzed at 16 weeks post transplantation to quantify CD45.2 chimerism and lineage contribution. For secondary transplantations, 3 × 106 cells of whole bone marrow was isolated and retransplanted 16 weeks post transplantation.Six hundred Dlk1+ , Dlk1− , or total HSCs –FITC, anti-CD45.2 (104)–PB, anti-CD11b (M1/70)-APCCy7, anti-GR1 (RB6.8C5)-APC, anti-CD8a (53.6.7)- PECy5, anti-CD4 (GK1.5)-PECy5, anti-B220 (RA3.6B2)-AF700.The CEL-Seq2 protocol with reduced volumes was used as previously described  and modified using the following reagents.Escherichia coli DNA polymerase I, E. coli DNA ligase, RNase H  and 5 × second-strand buffer were replaced with E. coli DNA polymerase , E. coli DNA ligase , RNaseH , and 10 × Second-Strand Buffer  respectively.Instead of 1.2 μl vapor lock as hydrophobic encapsulation barrier mineral oil  was used. For cDNA first-strand synthesis, Protoscript II and Protoscript II Reaction Buffer  as well as murine RNase-Inhibitor  was used instead of SuperScript II reverse transcriptase, first-strand synthesis buffer and RnaseOUT. The water volume was adjusted to adequately dilute the 10x second-strand buffer. After second-strand synthesis, 96 wells were pooled, which results in 96 single cells per library.The library preparation was performed as previously described , but by Paired-end reads were aligned to the transcriptome using BWA (version 0.6.2-r126) with default parameters . The traPPP1R2 and PDCD4 mRNAs were designed with the Stellaris RNA FISH probe designer  and produced by LGC Biosearch Technologies.Singly labelled oligonucleotides (Quasar 570 or Quasar 670) targeting g) to remove the supernatant. Briefly, naïve CD8 + T cells were washed once with PBS, fixed in 3.7% formaldehyde in PBS for 10 min at room temperature, and washed again twice with PBS. Cell pellet was resuspended in 200 μl of 70% ethanol, incubated at 4 °C for 1 h and then washed with 200 μl of wash buffer A  supplemented with 10% deionized formamide  at room temperature for 5 min. Cells were hybridized with 80 μl of hybridization buffer  supplemented with 10% deionized formamide containing the FISH probes at a 1:100 dilution at 37 °C overnight. The next day, cells were washed with 200 μl of wash buffer A supplemented with 10% deionized formamide at 37 °C for 30 min and stained with wash buffer A supplemented with 10% deionized formamide and 10 μg/ml Hoechst 33,342  at 37 °C for 30 min. Cells were rinsed once with 200 μl of 2 × SSC, equilibrated 5 min in base glucose buffer , and then incubated 5 min in base glucose buffer supplemented with a 1:100 dilution of glucose oxidase (stock 3.7 mg/ml) and catalase (stock 4 mg/ml). Cell pellet was resuspended in 10 μl of ProLong Glass Antifade Mountant  and mounted on a glass slide with a glass coverslip.Naïve CD8 + T cells were isolated from the peripheral blood of two healthy donors using the Naive CD8 + T Cell Isolation kit  according to the manufacturer’s instructions. SmRNA FISH procedure was performed in suspension, with brief centrifugations between steps  and FISH spots within nucleus and cytoplasm were quantified.Additional file 1: Supplementary figures and tables.Additional file 2. Review history


N-methylserotonin is reported as its hydrogen oxalate salt.The structure of the natural product  N-methylserotonin {systematic name: [2-(5-hydroxy-1H-indol-3-yl)ethyl](methyl)azanium hydrogen oxalate}, C11H15N2O+·C2HO4−, is reported. The structure possesses a singly protonated N-methylserotonin cation and one hydrogen oxalate anion in the asymmetric unit. In the crystal, the molecules are linked by N—H⋯O and O—H⋯O hydrogen bonds into a three-dimensional network.The solid-state structure of  N-methylserotonin, (ii) 5-hydroxy-N,N-dimethyltryptamine (bufotenine) and (iii) 5-hydroxy-N,N,N-trimethyltryptammonium (bufotenidine). Of these, bufotenine is probably most widely known as a natural product found in the secretions of Bufo alvarius toads. Bufotenine is a potent agonist of serotonin receptors and is one of several compounds to which the psychedelic effects of toad secretions are attributed  is a ubiquitous neurotransmitter that is integral in regulating mood, anxiety and happiness in humans °. The hydrogen oxalate anion varies significantly from planarity, with a CO2-to-CO2 plane-to-plane twist angle of 24.2 (1)°. The ions are linked together through a series of N—H⋯O and O—H⋯O hydrogen bonds into a three-dimensional framework .Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314623003784/bx4024sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314623003784/bx4024Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314623003784/bx4024Isup3.cmlSupporting information file. DOI: 2259219CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


CDC42/RAC guanine nucleotide exchange factor, βPix, binds to β-catenin in colon cancer cells, augmenting β-catenin transcriptional activity. Using in silico, in vitro, and in vivo approaches, we explored whether these actions are regulated by M3R. At the invasive fronts of murine and human colon cancers, we detected co-localized nuclear expression of βPix and β-catenin in stem cells overexpressing M3R. Using immunohistochemistry, immunoprecipitation, proximity ligand, and fluorescent cell sorting assays in human tissues and established and primary human colon cancer cell cultures, we detected time-dependent M3R agonist-induced cytoplasmic and nuclear association of βPix with β-catenin. βPix knockdown attenuated M3R agonist-induced human colon cancer cell proliferation, migration, invasion, and expression of PTGS2, the gene encoding cyclooxygenase-2, a key player in colon neoplasia. Overexpressing βPix dose-dependently augmented β-catenin binding to the transcription factor TCF4. In a murine model of sporadic colon cancer, advanced neoplasia was attenuated in conditional knockout mice with intestinal epithelial cell deficiency of βPix. Expression levels of β-catenin target genes and proteins relevant to colon neoplasia, including c-Myc and Ptgs2, were reduced in colon tumors from βPix-deficient conditional knockout mice. Targeting the M3R/βPix/β-catenin axis may have therapeutic potential.M PTGS2, cyclooxygenase2 (COX2)]. In colon cancer cells, muscarinic receptor (MR) activation augments β-catenin signaling1. Of five MR subtypes, CHRM3, a conditional oncogene encoding M3R2, is overexpressed in 60–80% of colon cancers5. Post-M3R signaling selectively induces genes promoting cell proliferation, migration, and invasion13. Interconnecting mechanisms involving activation of protein kinase C-α (PKC-α) and transactivation of epidermal growth factor receptors (EGFR) mediate post-M3R signaling. M3R deficiency attenuates murine intestinal neoplasia14. Attenuated nuclear accumulation of β-catenin and intestinal tumor formation in M3R-deficient ApcMin/+ mice15, led us to explore functional crosstalk between M3R and β-catenin signaling.In quiescent intestinal epithelial cells, cytosolic β-catenin is sequestered in a multi-protein complex and targeted for degradation. In ~ 90% of colon cancers APC or β-catenin mutations destabilize β-catenin destruction complexes, freeing β-catenin to translocate to the cell nucleus. As a transcriptional coactivator of TCF4, nuclear β-catenin induces transcription of genes that promote neoplasia, e.g., prostaglandin-endoperoxide synthase 2   revealed frequent ARHGEF7 amplification in colorectal cancer  and 12 (71%) cancers, respectively; ARHGEF7 and CHRM3 were both over-expressed in seven of 17 cancers , and both βPix and M3R immunostaining were more intense in cancer compared to normal tissue /ERK pathway; there is extensive crosstalk between signaling pathways52. To explore post-receptor signaling underlying MR agonist-induced nuclear binding of βPix to β-catenin, we used previously validated chemical inhibitors52. Pre-treating HT-29 cells with Gö6976, a PKC inhibitor, or with AG-1478, an EGFR inhibitor, nearly abolished MR agonist-induced binding of βPix to β-catenin , using an antibody that recognizes activated β-catenin unphosphorylated at Ser-37 and Thr-41 residues55. Treating primary colon cancer cells with ACh stimulated striking cytoplasmic and nuclear colocalization of βPix and activated β-catenin . We used fluorescence cell sorting to explore if MR activation stimulated nuclear translocation of βPix or β-catenin. After treating HT29 cells with 200 µM ACh for 10 min, we measured shifts in nuclear localization of total β-catenin, activated β-catenin, and βPix. We failed to detect an increased nuclear signal for either total or activated β-catenin Fig. e, right.We were curious to see if β-catenin transcriptional activity modulated βPix expression. We verified that siRNA-treated HT-29 and SNU-C4 cells had reduced β-catenin expression; Fig. 3R agonists stimulate binding of pre-existing nuclear βPix to β-catenin and not cytoplasm-to-nucleus translocation of either βPix or β-catenin. Thus, our working model suggests M3R activation stimulates βPix binding to β-catenin independently within the cytoplasm and nucleus. We performed a pilot experiment to test whether nuclear βPix bound to β-catenin could participate as a TCF-4 transcriptional cofactor. We used anti-TCF-4 antibodies to determine whether exogenous Flag-tagged βPix augmented β-catenin binding to TCF-4. Immunoprecipitation assays using nuclear extracts derived from HCT116 colon cancer cells incubated with increasing concentrations of Flag-βPix revealed progressively augmented Flag-βPix and β-catenin binding to TCF-4 . We used the same approach to knock down βPix expression in HT-29 cells and confirmed ACh (100 μM for 24 h) did not alter βPix expression in naïve or transfected cells . Next, we examined the effects of stable knockdown of βPix expression on cell invasion. shRNA against ARHGEF7 robustly knocked down βPix expression in HT-29 cells; control shRNA had no effect  littermate controls with AOM/dextran sodium sulfate (DSS) and harvesting colons after 20 weeks  , a stronger effect of βPix deficiency than without MR agonist treatment .We explored the role of βPix in vivo by treating 30  eks Fig. a. Body weks Fig. b. Arhgefse) Fig. c − 55% fent Fig. a. ArhgefcMyc, Ccnd1, and Ptgs2 (Cox2)56, we compared expression of these genes in adenocarcinomas from CKO and control mice. mRNA expression levels for cMyc and Ccnd1, the genes encoding c-myc and cyclin D1 were reduced ~ 50% in tumors from Arhgef7 CKO mice . Removing an ‘outlier’ from the Ptgs2 dataset , uncovered 70% reduced Ptgs2 expression in βPix-deficient cancers .Having previously observed a relationship between MR activation and expression of Arhgef7 CKO and six control mice. Whereas β-actin loading controls were similar in gels from control and CKO mice, except for Cox2 signals in CKO mice 2 and 4, expression of colon cancer-related proteins was substantially reduced in βPix-deficient adenocarcinomas . We failed to detect a correlation between ARHGEF7 and PTGS2 mRNA expression , catalyzes the metabolism of arachidonic acid to prostaglandins and other bioactive molecules, must be carefully regulated. Elevated levels of COX2 are associated with the development and progression of colon neoplasia62 and reduced survival63; in both animal models and humans, inhibiting COX-2 activity with non-steroid anti-inflammatory drugs attenuates colon neoplasia. MR activation in human colon cancer cells up-regulates COX2 expression4. We found Ptgs2 expression was also greatly reduced in tumors from Arhgef7 CKO mice with intestinal epithelial cell-selective βPix deficiency. Finding a connection between the M3R/βPix/β-catenin and COX2 expression provides a mechanistic framework that may be clinically relevant. Indeed, the lack of a relationship between ARHGEF7 and PTGS2 expression in colon cancer suggests M3R activation is pivotal to augmenting COX2 expression by a βPix-dependent mechanism.In human colon cancer cells, we observed that βPix knockdown reduced PTGS2, important for cancer progression62. Reduced βPix expression attenuates β-catenin target gene expression, cell function, and murine colon tumor formation. Nonetheless, we acknowledge that our work has limitations. In vitro cell models may not recapitulate complex in vivo interactions and murine cancer models, while mimicking key features of human colon cancer progression, do not fully mirror the development and progression of human colon neoplasia. Lastly, a putative role for βPix as a cofactor in the β-catenin/TCF4 transcription factor complex must remain speculative until confirmed by complementary approaches. Pursuing this intriguing finding and confirming a functional interaction between endogenous βPix and the β-catenin/TCF-4 transcription factor complex that alters β-catenin target gene expression requires luciferase reporter, electrophoretic mobility shift, chromatin immunoprecipitation, and other approaches beyond the scope of the current project and will be pursued in future work.Based on the current findings, we propose a model wherein post-MR signal transduction, mediated primarily by PKC-α activation, stimulates βPix binding to β-catenin in the cytoplasm and nucleus. Our preliminary data suggest nuclear βPix binds β-catenin/TCF4 transcription complexes, augmenting key β-catenin target gene expression, e.g., 3R and β-catenin signaling that modulates colon cancer gene expression and cell function. Employing rigorous in vitro and in vivo experimental designs to test established human colon cancer cell lines and fresh human colon cancer cells using complementary innovative approaches, we uniformly observed M3R agonist-stimulated βPix/β-catenin binding in both the cytosolic and nuclear compartments and consistent effects on downstream expression of β-catenin target genes. Targeting the M3R/βPix/β-catenin axis has therapeutic potential. Our experience using Arhgef7 CKO mice with intestinal epithelial cell-selective βPix deficiency suggests targeting intestinal βPix would be safe; Arhgef7 CKO mice are healthy and fertile with modest phenotypic changes59. Lastly, since other upstream receptors act through PKC-α, our findings may be mechanistically relevant to other receptors and signaling pathways in colon neoplasia and other cancers in which muscarinic receptor signaling plays a prominent role.In conclusion, our findings identify a noncanonical role of βPix as an intermediary between MChemicals and reagents were obtained from the following sources: Matrigel from Corning #356,231), azoxymethane (AOM) from MRIGlobal (#0061); and dextran sodium sulfate (DSS) from MP Biomedical . If not otherwise specified, chemicals and reagents were from Sigma-Aldrich, St. Louis, MO, USA. Antibodies were obtained and used as follows: anti-βPix antibodies from Millipore , Santa Cruz , and Sigma-Aldrich ; anti-total β-catenin from Abcam (#ab19381); anti-activated-β-catenin from Millipore (05–665-AF555); Alexa Fluor 488 from Invitrogen (#A21206); Alexa Fluor 594 from Invitrogen (#A21203); anti-GAPDH from Cell Signaling (#2118); anti-FLAG antibody from Sigma-Aldrich (#F1365); and anti-Ki67 from Bethyl Labs (#IHC-00375). PCR primers are shown in Supplemental Table , azoxymeArhgef7flox/flox-/Tg(Villin-Cre) mice (Arhgef7 CKO mice) with intestinal epithelial βPix deficiency59. Mice were bred, housed, and treated under identical conditions in a pathogen-free room. To achieve microbiome equivalence, we co-housed animals throughout these studies until they were euthanized. Experiments were approved by the Institutional Animal Care and Use Committee of the University of Maryland, Baltimore, and VA Maryland Health Care System Research and Development Committee. All experiments were performed in accordance with institutional guidelines and regulations and the authors complied with the ARRIVE guidelines.We created 2 in a humidified incubator and passaged weekly at subconfluence after trypsinization.HT-29, H508, SNU-C4, and HCT116 human colon cancer cell lines were authenticated and purchased from American Type Culture Collection. HT-29 and HCT116 cells were grown in McCoy's 5A medium and H508 and SNU-C4 wells were grown in RPMI 1640 (Life Technologies-Thermo Fisher), both supplemented with 10% FBS. Cells were grown at 37ºC, with 5% CO64, after washing (HBSS), we cut tissues into 1–2 mm3 sections, and placed them in 0.1% trypsin at 4 °C overnight. We then placed tissues in complete growth media (RPMI 1640 supplemented with 10% FBS with an antibiotic–antimycotic) and incubated the material at 37 °C for 15 min. We then washed tissues (HBSS) before additional incubation in 0.1% collagenase for 45 min at 37 °C (5% CO2). After filtration (40-μm filters), we centrifuged cells at 150 × g for 5 min at room temperature, resuspended cells in culture media, and seeded cells in T25 flasks.We collected deidentified discarded fresh tissue samples from colon adenocarcinomas and adjacent normal colon from patients undergoing primary colon cancer resection at the University of Maryland Medical Center . As described previously65. Briefly, we grew fresh primary cancers on slides, fixed in cold MeOH for 10 min and permeabilized with 0.1% Triton-X-100 for 10 min on ice. We incubated cells with blocking solution for 60 min and incubated overnight (4 °C) with anti-βPix and -β-catenin antibodies. We then incubated cells with PLA probes for 1 h, with ligation solution for 30 min, and amplification solution for 100 min . To identify cell nuclei, we stained slides with 4',6-diamidino-2-phenylindole (DAPI). We acquired images using an LSM 510 inverted confocal microscope; dots in the cell nucleus represented βPix molecules within 40 nm of β-catenin molecules.We used the Duo-link In Situ-Fluorescence kit following both the manufacturer's instructions (Sigma-Aldrich) and published methods59, we synthesized first-strand cDNAs from 5 µg RNA  and performed qPCR using 50 ng cDNA, the SYBR Green PCR Master Mix (Applied Biosystems), and 0.5 μM in 20 μl forward and reverse primers. We designed primers . We performed qPCR using Step One (Applied Biosystems) with Power SYBR Green Master Mix (ABI). PCR conditions were as follows: 5 min at 95 °C followed by 37 cycles of 95 °C for 15 s, 60 °C for 20 s, 72 °C for 40 s, and final cycles at 95 °C for 15 s, 60 °C for 15 s, and 95 °C for 15 s. We normalized gene expression to β2-microglobulin (B2M) and analyzed qPCR data using the comparative CT (2C–ΔΔT) method.As described previously38, lysed cells in 20 mM Tris, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1% Triton X-100, 1 mM sodium fluoride, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin, and 1 µg/ml leupeptin. We separated equal amounts of protein by 7.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes (Millipore), immunoblotted, and visualized bands by chemiluminescence (Amersham Biosciences). For immunoprecipitation, we added antibodies against β-catenin or βPix to cytoplasmic or nuclear lysates for 2 h, followed by protein A- or protein G-agarose beads for 1 h. We washed beads thrice in PBS, released immunoprecipitated proteins from beads by boiling in 1 × sample buffer (5 min), and immunoblotted. We employed β-actin and H2A loading controls to ascertain that equal amounts of cytoplasmic and nuclear proteins, respectively, were included in the extracted protein input. After immunoprecipitation, as a second loading control, we immunoblotted for expression of the bait protein ; this is marked by a vertical line to the left of the images obtained from the same gel.For immunoblotting, we treated cells with MR agonists and antagonists at the concentrations and times indicated and, as described previously38, we washed cells twice with ice-cold PBS, harvested cells with a rubber policeman, and lysed cells in 20 mM HEPES, pH 7.0, 10 mM KCl, 2 mM MgCl2, 0.5% Nonidet P-40, 1 mM Na3VO4, 10 mM NaF, 1 mM phenylmethanesulfonyl fluoride, and 2 µg/ml aprotinin. After a 10-min incubation on ice, we homogenized cells (20 strokes in a Dounce homogenizer) and sedimented nuclear homogenates by centrifuging at 1,500 × g for 5 min. Supernatants were centrifuged at 16,000 × g for 20 min; the resulting supernatant was the non-nuclear fraction. To remove contaminating cytoplasmic membranes, we washed nuclear pellets thrice with lysis buffer. To extract nuclear proteins, we resuspended isolated nuclei in 150 mM NaCl, 1 mM EDTA, 20 mM Tris–Cl, pH 8.0, 0.5% Nonidet P-40, 1 mM Na3VO4, 10 mM NaF, 1 mM phenylmethanesulfonyl fluoride, and 2 µg/ml aprotinin, and sonicated the material for nuclear lysis. We collected nuclear lysates after centrifugation  and electrophoresed the material on 7.5% SDS–polyacrylamide gels. We transferred proteins to polyvinylidene difluoride membranes, immunoblotted with antibodies, and detected bands by electrochemiluminescence.As described previously38.We performed FLAG-βPix and transient transfection of HCT116 colon cancer cells with FLAG-βPix using Trans-It transfection 2020 reagent  following manufacturer’s instructions66. We transfected HCT116 cells with siRNA targeting human βPix: siRNA #1, 5’-GAGCUCGAGAGACACAUGGTT-3’; siRNA #2, 5’-GGAUAUUAGUGUCGUGCAATT-3’ (Ambion)38. Silencer Negative Control siRNA #1 (Invitrogen) was used as control. For shRNA experiments using HT-29 cells, we targeted human GIPZ viral particles against the following sequence: AGGATGAAGTTCAAGAATT (Thermo Scientific)38. For shRNA, we used non-silencing-GIPZ lentiviral shRNAmir (Thermo Scientific) as control.We transfected HT-29 and SNU-C4 cells with 50 nM siRNA targeting human β-catenin67, we seeded HT-29 cells (4.5 × 106 cells) in 60-mm plates for 24 h. We serum starved cells for 24 h before adding 200 µM ACh for 10 min. After trypsinization, we fixed cells with 1% PFA on ice for 15 min and filtered them through 40-µm filters. We resuspended cells in 80% ethanol, incubated them for 4 min on ice, and washed with PBS. We blocked cells in PBS with 7% donkey serum and 0.1% TX-100 for 30 min at room temperature before adding primary anti-β-Pix and anti-activated β-catenin antibodies for 1 h at 4 °C. After washing with PBS, we incubated cells with secondary antibody . We washed cells with PBS, stained nuclei with DAPI at room temperature (15 min) and washed again with Ca2+/Mg2+-free DPBS. We performed flow cytometry and measured intracellular fluorescence . We collected bright field and fluorescent images  at a × 60 magnification. To determine nuclear localization of β-Pix (AF488 attached) and activated β-catenin (AF555 attached), we analyzed 5,000 gated cell singlets per sample and used IDEAS Analysis Software (Amnis) and the Similarity Morphology Feature.Applying methods described previously3 cells per well and transfected cells with 50 nM control or βPix siRNA using TransIT-siQuest reagent according to the manufacturer (Mirus). After 24 h, we stimulated cells with 100 µM ACh for 48 h and measured cell proliferation using the CellTiter-Glo assay kit (Promega). For cell migration assays, we placed HT-29 cells in the upper chamber of uncoated inserts (BD Biosciences). Cells on the underside of inserts were fixed, stained , and counted in five random high-power fields (HPF). We performed Invasion assays using Biocoat Matrigel Invasion Chambers (Corning) as described previously52.For cell proliferation assays, we seeded HCT116 cells at 7 × 10Arhgef7 CKO and littermate control male and female mice with once weekly intraperitoneal injection of 7.5 mg AOM/kg body weight for four weeks. Starting one week after the last AOM injection, 2.5% DSS was added to the drinking water for 5 days. In a separate experiment, after AOM/DSS treatment, we added bethanechol (400 µg/ml) to the drinking water for 15 weeks. Twenty weeks after the first AOM injection, investigators masked to genotype euthanized mice, harvested colons, and measured tumor number and size.We treated 10- to 17-week-old Oncomine™ (www.oncomine.org)44 to compare gene mRNA levels in colon cancer and normal adjacent tissue. GEPIA (gepia.cancer-pku.cn) analyzes RNA expression in tumor and normal samples from the National Cancer Institute Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases45. The Human Protein Atlas Program provided information on proteins in cells, tissues, and organs46 and used UALCAN  to analyze OMICS data47. We compared ARHGEF7, CHRM3, and PTSG2 mRNA expression levels as described57.We used t-test, Mann–Whitney U test, one-way ANOVA, with either Tukey’s HSD post hoc or Dunn’s tests using SigmaPlot 13.0  and two-tailed Fisher’s exact test using GraphPad Prism . We considered a p-value < 0.05 statistically significant.Unless indicated otherwise, we expressed data as mean ± SE from at least three experiments and analyzed results using Student’s two-tailed Supplementary Information.


In the crystal structure, the complex cation exhibits two square-planar RhI centres, each bound by a cyclooctadiene (COD) ligand and by two adjacent imidazolylidene N-heterocyclic carbene (NHC) donors from the same phenoxy ring of the {tetrakis(methylene)}tetrakis(1-methyl-1H-imidazol-2-yl) (L) ligand. The dibenzo-crown ether bridge of L spans the Rh centres and forms hydrogen bonds with water molecules. One water molecule with half occupancy bridges adjacent macrocycles in the lattice. Another water with full occupancy forms weak hydrogen bonds to the crown ether O atoms and is, in turn, part hydrogen bonded by a lattice water with half occupancy. The latter water is within hydrogen-bonding distance of a fourth water also with partial occupancy. The result of these interactions is the formation of a layer in the ab plane. Two PF6− ions, one of which is twofold disordered, and one ordered and one twofold disordered (with 0.5 occupancy) lattice acetonitrile molecules complete the crystal structure.The compound (μ-3,3′,3′′,3′′′-{[2,5,8,15,18,21-hexaoxatricyclo[20.4.0.0 L){Rh(COD)}2][PF6]3, which has a potassium ion bound within the crown ether bridge of the ligand L, see: Shrestha et al. (20112(COD) centres, see: Mata et al. 2(C40H42N8O6)](PF6)2·1.5C2H3N·2H2O = 0.051wR(F2) = 0.164S = 1.3311473 reflections964 parameters270 restraintsH atoms treated by a mixture of independent and constrained refinementmax = 1.15 e Å−3Δρmin = −1.40 e Å−3ΔρBLU-ICE  used to solve structure: SHELXS97  I, global. DOI: Click here for additional data file.10.1107/S160053681204901X/tk5170Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


In one of the cations, the three symmetry-unique O—H groups are disordered over two sets of sites with refined occupancy ratios of 0.701 (9):0.299 (9), 0.671 (8):0.329 (8) and 0.566 (7):0.434 (7). In the anion, the ZnII ion is coordinated in a distorted tetrahedral environment by four S atoms of two chelating 1,3-dithiole-2-thione-4,5-dithiolato ligands. The dihedral angle between the mean planes [maximun deviations = 0.022 (3) and 0.0656 (6) Å] of the two ligands is 87.76 (3)°. An intamolecular O—H⋯O hydrogen bond occurs in the disordered cation. In the crystal, O—H⋯O and O—H⋯S hydrogen bonds link the components into a two-dimensional network parallel to (0-11).In the asymmetric unit of the title compound, (C DOI: 10.1107/S1600536813014992/lh5618Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


T, a 2-chloro-4-nitrophenol (2C4NP) degrading bacterium, was isolated from soil collected from a pesticide-contaminated site in Punjab, India. The strain, which stained Gram positive, displayed a rod-coccus life cycle, and possessed a type A3α peptidoglycan (l-Lys–l-Ala3), MK-9(H2) as the major menaquinone, anteiso-C15 and iso-C15:0 as the major cellular fatty acids, and diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and a glycolipid as the major polar lipids, showed morphological and chemotaxonomic properties consistent with those reported for members of the genus Arthrobacter. Phylogenetic analysis of the 16S rRNA gene sequence of strain SJConT confirmed that it was a member of this genus with Arthrobacter globiformis DSM 20124T being the closest relative (sequence similarity of 97 %). The DNA G + C content of strain SJConT was 69 ± 1 mol% and DNA homology with A. globiformis DSM 20124T was 45 %, suggesting that strain SJConT represented a novel species of the genus Arthrobacter, which we have named Arthrobacter nitrophenolicus sp. nov The type strain is SJConT (=MTCC 10104T =DSM 23165T).Strain SJCon Arthrobacter was first proposed by Conn and Dimmick   .The primary habitat of T from a pesticide-contaminated soil by an enrichment method using 2-chloro-4-nitrophenol as sole carbon source  has been reported previously  at ×1000 and motility was checked using the method described by Skerman . Gram stT was streaked on nutrient agar plates, incubated at different temperatures and the growth on the plate scored. The growth in nutrient broth containing different NaCl concentrations (0.5–7 %) was monitored by measuring optical density at 600 nm in a Lambda 35 spectrophotometer (Perkin Elmer). Growth at different pH (pH 4.5–12) was tested in nutrient broth after the pH had been adjusted using appropriate buffers as described previously  was determined using Nutrient agar plates. Strain SJCon2S and indole, reduction of nitrate and acid production from carbohydrates were determined as described previously . For this, the inoculum was prepared by re-suspending Nutrient agar grown colonies to a turbidity equivalent to 0·5 McFarland units and the GP2 Microplates inoculated following the manufacturer’s instructions. The plates were incubated at 30 °C for 24 h and the results read using a MicroPlate Reader equipped with Microlog 4.2 software. The results are listed in the species description.2), a characteristic chemotaxonomic marker of the genus Arthrobacter. The fatty acid profile comprises C16:0 (6.48 %), C18:0 (1.61 %), iso-C14:0 (0.93 %), iso-C15:0 (16.33 %), anteiso-C15:0 (44.18 %), iso-C16:0 (9.25 %), iso-C17:0 (5.61 %) and anteiso-C17:0 (15.19 %). The major polar lipids were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylinositol (PI) and a glycolipid. The peptidoglycan was identified as type A3α (l-Lys–l-Ala3).Menaquinones, fatty acids, polar lipids and peptidoglycan were analyzed by standard methods   using the thermal denaturation (Tm) method and determined to be 69 ± 1 mol%.T with A. globiformis DSM 20124T was found to be 45 %.DNA hybridization was performed using Biotin DecaLabelTM Kit and Biotin Chromogenic Detection Kit (Fermentas Life Sciences) following the manufacturer’s instruction. The DNA–DNA relatedness value of strain SJConT degrades 2-chloro-4-nitrophenol, 4-nitrophenol and 3-methyl-4-nitrophenol. It possesses morphological characteristic of rod-coccus life cycle, which is consistent with that reported for members of the genus Arthrobacter. It’s affiliation to this genus is supported by the chemotaxonomic traits and 16S rRNA phylogeny. In addition, the presence of cell-wall peptidoglycan type A3α and menaquinone MK-9(H2) supports the placement of strain SJConT within the “globiformis” group. However, the large phylogenetic distance, the low DNA homology and the higher mol % G + C content and a number of phenotypic traits (Table strain SJConT from A. globiformis DSM 20124T and members of the “globiformis” group. The fatty acid composition of strain SJconT was differed from the fatty acid composition of A. globiformis DSM 20124T (Table T was comprises C16:0 (6.48 %), C18:0 (1.61 %), iso-C14:0 (0.93 %), iso-C15:0 (16.33 %), anteiso-C15:0 (44.18 %), iso-C16:0 (9.25 %), iso-C17:0 (5.61 %) and anteiso-C17:0 (15.19 %). On the basis of these polyphasic differences, strain SJConT represents a novel species of the genus Arthrobacter, for which the name Arthrobacter nitrophenolicus sp. nov. is proposed. The type strain is SJConT (=MTCC 10104T =DSM 23165T).Strain SJConNitro containing nitrogen, N.L. n. phenol phenol; M.L. adj. nitrirophenolicus relating to nitrophenol)., C18:0 (1.61 %), iso-C14:0 (0.93 %), iso-C15:0 (16.33 %), anteiso-C15:0 (44.18 %), iso-C16:0 (9.25 %), iso-C17:0 (5.61 %) and anteiso-C17:0 (15.19 %) are the major fatty acids. The major polar lipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and a glycolipid. The major menaquinone detected is MK-9(H2) and the peptidoglycan-type is A3α. The DNA G + C content of strain SJConT is 69 ± 1 mol%.Cells are Gram-positive, catalase-positive, oxidase-negative, non-spore-forming, non-motile and exhibit a rod–coccus growth cycle. The optimal temperature for growth is 30 °C (temperature growth range of 10–40 °C) and pH 7.0 (pH growth range of pH 6–10). The cells grow on nutrient broth medium with 4 % NaCl, and degrade 2-chloro-4-nitrophenol, 4-nitrophenol and 3-methyl-4-nitrophenol. Chlorohydroquinone, a major intermediate product of degradation of 2-chloro-4-nitrophenol, which is further degraded via formation of maleylacetate. It hydrolyses gelatin and casein but not starch. No growth occurs on Simmon Citrate and Mac Conkey Agar. Chlorohydroquinone is negative for indole production, HT =MTCC 10104T =DSM 23165T was isolated from a pesticide-contaminated soil sample collected in Punjab State, India.The type strain SJCon


Aβ perturbs a number of synaptic components including nicotinic acetylcholine receptors containing α7 subunits (α7-nAChRs), which are abundantly expressed in the hippocampus and found on GABAergic interneurons. We have previously demonstrated the existence of a novel, heteromeric α7β2-nAChR in basal forebrain cholinergic neurons that exhibits high sensitivity to acute Aβ exposure. To extend our previous work, we evaluated the expression and pharmacology of α7β2-nAChRs in hippocampal interneurons and their sensitivity to Aβ.GABAergic interneurons in the CA1 subregion of the hippocampus expressed functional α7β2-nAChRs, which were characterized by relatively slow whole-cell current kinetics, pharmacological sensitivity to dihydro-β-erythroidine (DHβE), a nAChR β2* subunit selective blocker, and α7 and β2 subunit interaction using immunoprecipitation assay. In addition, α7β2-nAChRs were sensitive to 1 nM oligomeric Aβ. Similar effects were observed in identified hippocampal interneurons prepared from GFP-GAD mice.These findings suggest that Aβ modulation of cholinergic signaling in hippocampal GABAergic interneurons via α7β2-nAChRs could be an early and critical event in Aβ-induced functional abnormalities of hippocampal function, which may be relevant to learning and memory deficits in AD. Aβ accumulation is considered to be a hallmark of Alzheimer’s disease (AD) and responsible for synaptic deficits and neuronal degeneration in AD . Althoug1-40 and Aβ1-42 to α7-nAChRs. Two other groups subsequently reported direct and functionally-relevant interactions of Aβ1-42 with α7-nAChRs . Perforated-patch whole-cell recordings coupled with a three-barrel drug application system were used . To prepare for perforated-patch whole-cell recording, glass microelectrodes  were fashioned on a two-stage vertical pipette puller , and the resistance of the electrode was 4–6 MΩ when filled with the internal solution. A tight seal (>2 GΩ) was formed between the electrode tip and the cell surface, which was followed by a transition from on-cell to whole-cell recording mode due to the partitioning of amphotericin B  into the membrane underlying the patch. After whole-cell, an access resistance lower than 60 MΩ was acceptable for perforated-patch recordings under voltage-clamp mode. The series resistance was not compensated in the experiments using dissociated neurons. Data were acquired by Axopatch 200B amplifier at 5 kHz with pClamp 9.2 software  and analyzed with Clampfit 9.2 software .Neuron dissociation and patch-clamp recordings were performed as described by Wu et al. ,30,67. B1-42, and Aβ1-42 . Aβ1–42 was reconstituted in distilled water to a concentration of 100 μM and stored at −80°C as previously described  containing 1× general protease inhibitor cocktails . The lysates were transferred to microcentrifuge tubes and further solubilized for 30 min at 4°C. The detergent extracts (supernatants) were collected by centrifugation at 15,000 × 1–42 peptides were analyzed with electrophoresis to test the exact form of oligomer during aggregation state. Pre-cast 10-20% SDS-polyacrylamide Tris-Tricine gels  or 16% Tris-Tricine gels in the presence or absence of SDS or Urea 8M were used. 100 μg of Aβ1-42 per sample was resuspended with 4X Tricine loading buffer. Aβ1-42 samples dissolved with water or DMSO were aggregated for 2 hr before loaded.Aβ p values less than 0.05 were considered statistically significant.All data were presented as mean ± standard error (SE). Statistical comparisons using Student’s t-test (independent or paired) were performed with Origin 5.0 . nAChRs: nicotinic acetylcholine receptors; Aβ: amyloid β peptide; DHβE: dihydro-β-erythroidine; AD: Alzheimer’s disease.The authors declare that they have no competing interests.QL: Performed experiments, analyzed data, and wrote manuscript. YH: Performed experiments using immunoprecipitation, and revised manuscript. JXS: Designed experiments and revised manuscript. SS: Designed experiments and revised manuscript. JW: Designed experiments, performed some patch-clamp recordings and wrote manuscript. All authors read and approved the final manuscript.


Insight into the T cell receptor (TCR) repertoire of effector T cells driving recurrent autoimmunity would aid the development of immunotherapies to prevent islet graft rejection. Accordingly, we used a multi-parameter flow cytometry strategy to assess the TCR variable β (Vβ) chain repertoires of T cell subsets involved in autoimmune-mediated rejection of islet grafts in diabetic NOD mouse recipients. Naïve CD4+ and CD8+ T cells exhibited a diverse TCR repertoire, which was similar in all tissues examined in NOD recipients including the pancreas and islet grafts. On the other hand, the effector/memory CD8+ T cell repertoire in the islet graft was dominated by one to four TCR Vβ chains, and specific TCR Vβ chain usage varied from recipient to recipient. Similarly, islet graft- infiltrating effector/memory CD4+ T cells expressed a limited number of prevalent TCR Vβ chains, although generally TCR repertoire diversity was increased compared to effector/memory CD8+ T cells. Strikingly, the majority of NOD recipients showed an increase in TCR Vβ12-bearing effector/memory CD4+ T cells in the islet graft, most of which were proliferating, indicating clonal expansion. Importantly, TCR Vβ usage by effector/memory CD4+ and CD8+ T cells infiltrating the islet graft exhibited greater similarity to the repertoire found in the pancreas as opposed to the draining renal lymph node, pancreatic lymph node, or spleen. Together these results demonstrate that effector/memory CD4+ and CD8+ T cells mediating autoimmune rejection of islet grafts are characterized by restricted TCR Vβ chain usage, and are similar to T cells that drive destruction of the endogenous islets.Islet transplantation provides a “cure” for type 1 diabetes but is limited in part by recurrent autoimmunity mediated by β cell-specific CD4 Our earlier work analyzing TCR Vα and Vβ gene usage by Major Histocompatibility Complex (MHC) class I tetramer-sorted CD8+ T cells specific for islet-specific glucose-6-phosphatase catalytic subunit-related protein derived peptide (IGRP206–214) indicated that islet graft destruction was mediated by clonotypes also prevalent in the pancreas of the diabetic NOD recipients + Teff has yet to be established. Furthermore, the clonotypic composition of β cell-specific CD4+ Teff mediating islet graft destruction has not been defined. Due to the several known and potential unknown autoantigens driving T1D, analysis of β cell-specific T cell populations by tetramer analysis is cumbersome and impractical to address these key issues.Islet transplantation is one approach to replace β cells and restore euglycemia in T1D patients + and CD8+ T cells infiltrating grafted and endogenous islets in individual diabetic NOD mice. The approach is advantageous since TCR Vβ usage can be readily assessed for different subsets of CD4+ and CD8+ T cells residing in multiple tissues of an individual animal. Herein we show that both CD4+ and CD8+ effector/memory T cells (Teff/mem) infiltrating an islet graft and the pancreas exhibit restricted TCR Vβ usage. Notably, whereas TCR Vβ usage by islet graft-infiltrating CD8+ Teff/mem is highly variable among individual animals, TCR Vβ12 is preferentially expressed by CD4+ Teff/mem in the majority of NOD recipients. Importantly, TCR Vβ usage by CD4+ and CD8+ Teff/mem is most similar between grafted and endogenous islets compared to peripheral lymphoid tissues in an individual NOD recipient. These results suggest that immunodominant β cell-specific T cells attacking the endogenous pancreas also mediate islet graft destruction.Accordingly, we have employed a novel multi-parameter flow cytometry approach to determine the TCR Vβ usage by CD4+ and CD8+ T cells stained with the anti-Vβ antibody panel used , CD44 and CD62L to identify eff/mem (CD44hi CD62Llo) and naïve (CD62Lhi CD44lo) T cell subsets + T cells in the analyses by intracellular staining of Foxp3  draining the graft site, pancreas, pancreatic lymph node (PLN), and spleen of individual recipients.Diabetic NOD female mice (blood glucose levels >250 mg/dL) were implanted with 500 syngeneic NOD.+ and CD4+ T cells, which represents the T cell repertoire under homeostasis, was essentially identical among the NOD recipients, and similar to the PLN and RLN  and endogenous  islets. These results demonstrate that TCR Vβ usage by naïve CD4+ and CD8+ T cells is diverse, with minimal (if any) variability among the tissues including grafted and endogenous islets in a given animal, and among NOD recipients.TCR Vβ usage by naïve splenic CD8 and RLN . Interesd RLN  , Vβ5.1/2 (19.5%), Vβ2 (10.9%), and Vβ8.1/2 (10.3%)  . Further, 9, 11)  comparedet graft , and 2)  T cells . General (10.3%) ; in the  (10.3%) . These f+ Teff/mem infiltrating the grafted and endogenous islets was increased compared to splenic naïve CD8+ T cells in the majority (7/13) of NOD recipients  was determined using H2Kd-IGRP tetramers. Strikingly, up to 65% of TCR Vβ8.1/2-expressing CD8+ Teff/mem in the grafted islets stained with the H2Kd-IGRP  of the majority of NOD recipients compared to naïve CD4+ T cells and CD4+ Teff/mem in the spleen  and endogenous (3.9±3.3%) islets relative to CD4+ Teff/mem found in the spleen (−0.1±0.8), RLN (2.0±1.6%) and/or PLN (−0.3±0.6%)  , 6A. Whipulation , 4, the pulation , 6A. In  T cells .+ Teff/mem may be explained by tissue-specific expansion. To address this possibility, the proliferative status of TCR Vβ12-expressing CD4+ Teff/mem was examined by measuring Ki67 expression + Teff/mem in islet grafts were Ki67+ (+ TCR Vβ12-expressing CD4+ Teff/mem was significantly increased (∼2.5-fold) in the islets, and to a lesser extent in the pancreas, compared to the corresponding splenic CD4+ Teff/mem , indicating a similar level of TCR Vβ diversity. In contrast, the entropy for TCR Vβ chains of CD8+ Teff/mem was reduced in the pancreas, and significantly lower in islet grafts compared to spleen, PLN and RLN  versus CD8+ (2.03±0.02) Teff/mem was significantly increased . These data demonstrate that CD8+ Teff/mem in the islet graft, and to a lesser extent the pancreas, have significantly restricted TCR Vβ chain usage in comparison to CD8+ Teff/mem in spleen and lymph nodes. The TCR repertoire of CD4+ Teff/mem in the grafted (and endogenous) islets, however, is more diverse than that of CD8+ Teff/mem.To more accurately assess the level of diversity among TCR Vβ chains expressed by naïve T cells and Teff/mem in the respective tissues, the Shannon entropy was calculated. The Shannon entropy within a given population represents both the species richness (number of Vβ chains expressed) and relative abundance (proportion of each Vβ chain) in the sample  and RLN , thereby and RLN . Further+ and CD8+ Teff/mem was similar between the islet graft and pancreas in individual recipients . Notably, the average divergence index detected for CD8+ Teff/mem in the islet graft versus pancreas (0.11±0.05) was reduced relative to comparisons between the islet graft and the spleen (0.18±0.03), PLN (0.22±0.03), and RLN (0.24±0.03) (+ Teff/mem where the divergence index for the TCR repertoire between the islet graft and pancreas (0.059±0.02) was less than the divergence index calculated for comparisons between the islet graft versus spleen (0.14±0.03), PLN (0.15±0.03) and RLN (0.13±0.03)  . Similar13±0.03) . These r+ and CD8+ T cells involved in autoimmune destruction of islet grafts in individual NOD recipients. The approach permits analyses of general shifts in TCR usage in a tissue-specific manner while taking in consideration the activation status of the T cells. Exploiting this strategy, 3 key observations were made. First, naïve CD4+ and CD8+ T cells make up a significant frequency of islet graft-infiltrating T cells, which express a TCR repertoire analogous to naïve T cells in peripheral lymphoid organs and endogenous islets. Second, TCR Vβ usage by islet graft-infiltrating Teff/mem is restricted, with the TCR repertoire of CD8+ versus CD4+ Teff/mem exhibiting distinct characteristics. Third, the TCR Vβ repertoire is similar for CD8+ and CD4+ Teff/mem infiltrating grafted and endogenous islets of a given NOD recipient.The TCR Vβ repertoire of islet-infiltrating T cells has been investigated mostly by RT-PCR from bulk RNA isolated from the islets or via flow cytometric-sorted MHC multimer-binding T cells + and CD8+ T cells are found at a relatively high frequency argues against antigen-specificity/stimulation playing an essential role for T cell trafficking into grafted and endogenous islets. This scenario is further supported by our observation that TCR Vβ usage by naïve T cells in the islet graft and pancreas is identical to the spleen, PLN and RLN  of NOD recipients . TCR Vβ usage by islet graft (and pancreas)-infiltrating CD4+ Teff/mem also differed in terms of the extent of overall diversity compared to CD8+ Teff/mem. Despite increased usage of certain TCR Vβ chains (e.g. Vβ12), islet graft-infiltrating CD4+ Teff/mem also expressed other Vβ chains at frequencies similar to naïve CD4+ T cells residing in the spleen (+ Teff/mem including Vβ2 (5.7%), Vβ4 (4.1%), Vβ6 (7.3%), Vβ8.3 (5.3%), Vβ10 (4.7%), and Vβ11 (4.2%)  . For instance, TCR Vβ12-expressing CD4 spleen) , consiststaining . Interesold less , which mt grafts , 5A. In + and CD8+ Teff/mem was more similar to the repertoire of Teff/mem residing in the pancreas than that detected in the PLN, RLN, and spleen  mice were bred and housed under pathogen-free conditions in an American Association for Laboratory-accredited animal facility. NOD mice were considered to be diabetic after 2 successive days of ≥250 mg/dl blood glucose as measured by a Freestyle Lite blood glucose monitor and strips (Abbott Diabetes Care Inc.). All procedures were reviewed and approved by the University of North Carolina Institutional Animal Care and Use Committee.NOD/LtJ and NOD.CB17.Prkdcscid) islets were transplanted under the renal capsule of the left kidney. Blood glucose values were monitored daily post-transplantation.Diabetic NOD female mice received 5 units of insulin daily prior to transplantation. Five hundred syngeneic (NOD.+ spleen cells (1×106) were added to wells containing cells from grafts and PLN/RLN prior to addition of antibodies as a staining internal control. Cells were stained with antibodies specific for CD90.2 (53–2.1), CD8 (53–6.7), CD44 (IM7), CD62L (MEL-14) (BD Biosciences), Thy1.1 (OX-7) (BioLegend), CD4+ (RM4-5) (Invitrogen) and three different anti-TCR Vβ panels. Panel A: αTCR Vβ2-biotin (B20.6), αTCR Vβ3-PE (KJ25), αTCR Vβ4-biotin (KT4), αTCR Vβ4-FITC, αTCR Vβ6-biotin (RRA-7), αTCR Vβ6-PE and αTCR Vβ9-FITC (MR10-2); Panel B: αTCR Vβ5.1/2-biotin (MR9-4), αTCR Vβ7-PE (TR310), αTCR Vβ8.1/2-FITC (MR5-2), αTCR Vβ 8.1/2-biotin and αTCR Vβ8.3-FITC (1B3.3); Panel C: αTCR Vβ10[b]-FITC (B21.5), αTCR Vβ 10[b]-PE, αTCR Vβ11-PE (RR3-15), αTCR Vβ11-biotin, αTCR Vβ12-biotin (MR11-1), αTCR Vβ13-PE (MR12-4) (Biolegend) and αTCR Vβ14-FITC (14–2). All αTCR Vβ antibodies were purchased from BD Biosciences unless noted. Binding of biotin-labeled antibodies was revealed by streptavidin Alexa 594 (Invitrogen). Cells were washed twice with PBS and stained with LIVE/DEAD® Fixable Blue Dead Cell Stain Kit (Invitrogen) to exclude dead cells. To stain for FoxP3 (FJK-16s) or Ki67 (B56) samples were washed, fixed and permeabilized with eBiosciences Fix/Perm kit following manufacturer’s indications. For tetramer analysis, H2Kd-IGRP206–214 tetramers were prepared as previously described d-IGRP206–214 for 40 minutes at room temperature, and then placed on ice and incubated for 20 minutes with 100 µL of a 2X cocktail of antibodies specific for T cell markers and TCR Vβ chains. Data was acquired at the University of North Carolina Flow Cytometry Facility using a 6 laser , 18 parameter LSRII Special Order Research Product flow cytometer (BD Biosciences). Analysis was performed with FlowJo software (Tree Star Inc.).Spleen, PLN, RLN, and pancreas single-cell suspensions were prepared by grinding tissue between frosted slides in RPMI complete containing 100 nM Dasatinib, which not only inhibits downregulation of the TCR but it also increases TCR and co-receptor surface expression allowing for better staining et alS is the total number of unique Vβ chains in the pool, and ip is the proportion of the pool represented by Vβ i, the Shannon entropy H is defined as:Shannon entropy was used as an index of TCR Vβ usage diversity. As described by Vincent 34 divergence was used as an index of similarity between TCR Vβ usage distributions and is defined as:ip is the proportion of the pool represented by Vβ i in the first sample and iq is the proportion of that Vβ in the second sample.The Kullback-LeiblerFigure S1+ and CD8+ Teff/mem was determined in the spleen, islet graft and pancreas of individual NOD recipients. ***p<0.001, **p<0.01; Student’s t test.Increased proliferation of islet graft-infiltrating Teff/mem in individual NOD recipients. The frequency of Ki67-staining CD4(TIFF)Click here for additional data file.


The anhydrous form can be prepared, however, and its structure is presented here. Similar to the hemihydrate form, the anhydrous phase contains the CoIII ion in an octahedral O2N4 coordination by a chelating carbonate group and four ammine ligands. The structure reveals an intricate array of N—H⋯O hydrogen bonds involving both the chelating and the non-chelating O atoms of the carbonate ligand as hydrogen-bond acceptors of the amine H atoms, which are also involved in hydrogen-bonding interactions with the nitrate O atoms. The structure of the anhydrous form is close to that of the hemihydrate phase, suggesting a probable topotactic reaction with relatively small rotations and translations of the [Co(CO3)(NH3)4]+ and NO3− groups during the dehydration process, which produces an unusual volume increase of 4.3%.Practical chemistry courses at universities very frequently propose the synthesis and characterization of [Co(CO For the crystal structure of the hemihydrate, see: Bernal & Cetrullo 1990; Junk & 3)(NH3)4]NO3[Co Åb = 6.7922 (5) Åc = 17.5394 (9) Åβ = 95.440 (3)°V = 931.21 (11) Å3Z = 4Kα radiation, λ = 1.5418 ÅCu T = 293 Kflat sheet, 8 × 8 mmSiemens D500 diffractometerSpecimen mounting: packed on the holderData collection mode: reflectionScan method: stepmin = 6.001°, 2θmax = 79.961°, 2θstep = 0.020°2θRp = 0.054Rwp = 0.072Rexp = 0.025RBragg = 0.030R(F) = 0.0262 = 8.123χ3751 data points114 parameters55 restraintsH atoms treated by a mixture of independent and constrained refinementDIFFRAC-AT  used to solve structure: ESPOIR  global, I. DOI: 10.1107/S1600536813017522/vn2075Isup2.rtvRietveld powder data: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


The 1,3-bis(imidazol-1-ylmethyl)benzene and 2,2′-dihydroxy-1,1′-methylenebis ligands connect the ZnII ions alternately in different directions, forming a layered structure parallel to the ac plane. Topological analysis reveals that the whole structure is a  network. The layers are further assembled into a three-dimensional supramolecular structure via C—H⋯O and C—H⋯π interactions.In the title compound, [Zn(C DOI: 10.1107/S1600536811054584/vm2140Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF reportAdditional supplementary materials:


The Ni—N bond lengths range from 2.074 (2) to 2.094 (2) Å. The dihedral angles between the three chelating NCCN groups to each other are 85.71 (3), 73.75 (2) and 85.71 (3)°, respectively. The Ni cation, the phenyl ring of the 1,10-phenanthroline ligand and the lattice water molecule are located on special positions (site symmetry 2). In the crystal, the uncoordinated 2,4,5-tricarboxybenzenoate anions join with each other through O—H⋯O hydrogen bonds, forming a two-dimensional hydrogen-bonded layer structure along the bc plane. The layers are further linked via additional O—H⋯O interactions between water and carboxyl groups, resulting in a three-dimensional supramolecular network.In the title compound, [Ni(C DOI: 10.1107/S1600536811048914/bq2318Isup2.hkl            Structure factors: contains datablock(s) I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials: