PJA1 mediates the effects of astrocytic GPR30 on learning and memory in female mice.

Hormone replacement therapy (HRT) is not recommended for treating learning and memory decline in menopausal women because it exerts adverse effects by activating classic estrogen receptors ERα and ERß. The membrane estrogen receptor G protein-coupled receptor 30 (GPR30) has been reported to be involved in memory modulation; however, the underlying mechanisms are poorly understood. Here, we found that GPR30 deletion in astrocytes, but not in neurons, impaired learning and memory in female mice. Astrocytic GPR30 depletion induced A1 phenotype transition, impairing neuronal function. Further exploration revealed that Praja1 (PJA1), a RING ubiquitin ligase, mediated the effects of astrocytic GPR30 on learning and memory by binding to Serpina3n, which is a molecular marker of neuroinflammation in astrocytes. GPR30 positively modulated PJA1 expression through the CREB signaling pathway in cultured murine and human astrocytes. Additionally, the mRNA levels of GPR30 and PJA1 were reduced in exosomes isolated from postmenopausal women while Serpina3n levels were increased in the plasma. Together, our findings suggest a key role for astrocytic GPR30 in the learning and memory abilities of female mice and identify GPR30/PJA1/Serpina3n as potential therapeutic targets for learning and memory loss in peri- and postmenopausal women.

Effectiveness and functional magnetic resonance imaging outcomes of Tuina therapy in patients with post-stroke depression: A randomized controlled trial.

Objective: To investigate the effectiveness and functional magnetic resonance imaging (fMRI) outcomes of Tuina therapy in patients with post-stroke depression (PSD). Methods: This was a single-center, randomized, two-armed, controlled trial. Eighty-four patients with PSD were selected and randomly assigned to a Tuina therapy group or a routine rehabilitation control group. The patients underwent five 20-min treatment sessions per week over a period of 2 weeks. The primary outcome measure was change in Hamilton Depression Rating Scale (HAMD) score over the 2 weeks of intervention, whereas the secondary outcome measures were changes in Fugl-Meyer Assessment (FMA) score, Modified Barthel index (MBI), and Mini Mental State Examination (MMSE) score. Results: The Tuina group showed significantly improved HAMD scores compared to the routine rehabilitation control group (5.85, [2.54, 9.16]). For the secondary outcomes, the Tuina group showed better MMSE scores than the routine rehabilitation group (1.97, [1.19, 2.76]); however, there were no significant differences between the other secondary outcomes of both groups (P > 0.05). After 2 weeks, both groups showed a significant decrease in HAMD score compared to baseline. In addition, the Tuina group showed a significant decrease in MMSE score compared to baseline (2.35, [1.8, 2.9]); however, there were no significant differences in the MBI and FMA scores of the two group after the intervention (P > 0.05). Regarding fMRI results, the zALFF values of the right caudate nucleus, right putamen, right insula, left superior temporal gyrus, right parahippocampal gyrus, right hippocampus, left middle temporal gyrus, left angular gyrus, and left thalamus were higher in the Tuina group. In the Tuina group, the functional connectivity between the hippocampus and thalamus, and the thalamus and caudate nucleus, were significantly different (P <0.01). In addition, the zALFF value of the hippocampus was significantly negatively correlated with HAMD score. No serious adverse events were observed in both groups. Conclusion: Tuina therapy administered 10 times within 2 weeks is safe and can effectively relieve depression and improve cognitive function in patients with PSD. This finding may be closely related to the effect of Tuina therapy on the activation and functional connectivity of the hippocampus. Clinical Trial Registration: http://www.chictr.org.cn/showproj.aspx?proj=55151, identifier ChiCTR200003388.

The efficacy of Tuina with herbal ointment for patients with post-stroke depression: study protocol for a randomized controlled trial.

BACKGROUND: Post-stroke depression (PSD) is a common complication after stroke which hinders functional recovery and return to social participation of stroke patients. Efficacy of conventional drug therapies for patients with PSD is still uncertain. Therefore, many patients prefer to use complementary and alternative therapies for PSD. Tuina (traditional Chinese manual manipulation) with herbal ointment is an integration of manual therapy, and ointment is an important part of traditional Chinese medicine (TCM) therapy. Preliminary experiments have shown that the Tuina with herbal ointment can improve the mental state of patients with PSD. The purpose of this study is to observe and verify the efficacy of Tuina combined with herbal ointment for patients with post-stroke depression, and to lay a foundation for further research on its mechanism of action. METHODS/DESIGN: In this study, a randomized controlled trial will be conducted in parallel, including two intervention groups: Tuina with herbal ointment group and herbal ointment for control group. A total of 84 eligible participants will be randomly assigned to the groups in a 1:1 ratio. All participants will receive conventional antidepressant venlafaxine treatment (75 mg QD), on which they received two different interventions. The interventions for both groups will be carried out 5 times each week for a period of 2 weeks. The primary outcome will be the Hamilton Rating Scale for Depression (HAMD). Secondary outcomes will include transcranial magnetic stimulation (TMS), as well as 36-item Short-Form Health Survey (SF-36) and Treatment Emergent Symptom Scale (TESS). They will be assessed at the baseline, at the end of the intervention (2 weeks), and during the 1 month and 3 months of follow-up by repeated measures analysis of variance. The significance level is 5%. Adverse events will be monitored at each visit to assess safety. All outcomes will be assessed and analyzed by researchers blinded to the treatment allocation. The purpose of this study will focus on observing the efficacy of Tuina with herbal ointment for patients with post-stroke depression, and to explore further the mechanisms of its effects. DISCUSSION: This study may evaluate clinical application value and safety of Tuina with herbal ointment in PSD patients, which can provide basis for clinical research and mechanism exploration of PSD. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR2000033887 . Registered on 15 June 2020. DISSEMINATION: The results will be published in peer-reviewed journals and disseminated through the study's website and conferences.

α-Lys424 Participates in Insertion of FeMoco to MoFe Protein and Maintains Nitrogenase Activity in Klebsiella oxytoca M5al.

Our previous investigation of substrates reduction catalyzed by nitrogenase suggested that α-Ile423 of MoFe protein possibly functions as an electron transfer gate to Mo site of active center-"FeMoco". Amino acid residue α-Lys424 connects directly to α-Ile423, and they are located in the same α-helix (α423-431). In the present study, function of α-Lys424 was investigated by replacing it with Arg (alkaline, like Lys), Gln (neutral), Glu (acidic), and Ala (neutral) through site-directed mutagenesis and homologous recombination. The mutants were, respectively, termed 424R, 424Q, 424E, and 424A. Studies of diazotrophic cell growth, cytological, and enzymatic properties indicated that none of the substitutions altered the secondary structure of MoFe protein, or normal expression of nifA, nifL, and nifD. Substitution of alkaline amino acid (i.e., 424R) maintained acetylene (C2H2) and proton (H+) reduction activities at normal levels similar to that of wild-type (WT), because its FeMoco content did not reduce. In contrast, substitution of acidic or neutral amino acid (i.e., 424Q, 424E, 424A) impaired the catalytic activity of nitrogenase to varying degrees. Combination of MoFe protein structural simulation and the results of a series of experiments, the function of α-Lys424 in ensuring insertion of FeMoco to MoFe protein was further confirmed, and the contribution of α-Lys424 in maintaining low potential of the microenvironment causing efficient catalytic activity of nitrogenase was demonstrated.

Evidence That the Pi Release Event Is the Rate-Limiting Step in the Nitrogenase Catalytic Cycle.

Nitrogenase reduction of dinitrogen (N2) to ammonia (NH3) involves a sequence of events that occur upon the transient association of the reduced Fe protein containing two ATP molecules with the MoFe protein that includes electron transfer, ATP hydrolysis, Pi release, and dissociation of the oxidized, ADP-containing Fe protein from the reduced MoFe protein. Numerous kinetic studies using the nonphysiological electron donor dithionite have suggested that the rate-limiting step in this reaction cycle is the dissociation of the Fe protein from the MoFe protein. Here, we have established the rate constants for each of the key steps in the catalytic cycle using the physiological reductant flavodoxin protein in its hydroquinone state. The findings indicate that with this reductant, the rate-limiting step in the reaction cycle is not protein-protein dissociation or reduction of the oxidized Fe protein, but rather events associated with the Pi release step. Further, it is demonstrated that (i) Fe protein transfers only one electron to MoFe protein in each Fe protein cycle coupled with hydrolysis of two ATP molecules, (ii) the oxidized Fe protein is not reduced when bound to MoFe protein, and (iii) the Fe protein interacts with flavodoxin using the same binding interface that is used with the MoFe protein. These findings allow a revision of the rate-limiting step in the nitrogenase Fe protein cycle.

Influence of blocking of 2,3-butanediol pathway on glycerol metabolism for 1,3-propanediol production by Klebsiella oxytoca.

Glycerol metabolism is a typical biological oxidoreductive reaction. 1,3-Propanediol (1,3-PD) is the final product of the reductive branch, while acetate, succinate, lactate, 2,3-butanediol (2,3-BD), and ethanol were produced in the oxidative branch. 2,3-BD, which has similar properties of high boiling point and water solubility with 1,3-PD, not only contests the carbon flow and NADH with 1,3-PD but also serves as an obstacle for obtaining high purity 1,3-PD in downstream processes. In this study, a 2,3-BD pathway-deficient mutant of Klebsiella oxytoca ZG36 was constructed by knocking out the budA gene of the wild-type strain M5al. The results of fed-batch fermentation by ZG36 indicated that the glycerol flux and the distribution of metabolites were altered in the K. oxytoca when the 2,3-BD pathway was blocked. No 2,3-BD was produced, and the activity of α-acetolactate decarboxylase (α-ALDC) can not be detected in the fermentation processes. The indexes of the 1,3-PD titer, the conversion from glycerol to 1,3-PD, and the productivity per cell dry weight (CDW) increased by 42%, 62%, and 46%, respectively, compared with the M5al, and the yield of the byproducts also increased obviously. The assay of the enzyme activities in the oxidative branch and the reductive branch of the glycerol metabolism, as well as the intracellular redox state, exposited the results logically.