The positive psychology of relational depth and its association with unconditional positive self-regard and authenticity

© 2020 World Association for Person-Centered & Experiential Psychotherapy & Counseling. Relational depth (RD) refers to moments in a therapeutic relationship in which a person has feelings of aliveness, satisfaction and immersion. However, no research has yet tested for the association between RD and concepts closely aligned with Carl Rogers’ hypothesis of how people change in a growth-promoting relationship. In this study, 55 therapy clients completed the relational depth inventory (RDI), the unconditional positive self-regard scale (UPSR) and the authenticity scale (AS). It was found that higher scores on the RDI were associated with higher scores on the UPSR and the AS. These results provide initial evidence for the growth-promoting effects of RD. Further prospective research is now warranted

GSK3B induces autophagy by phosphorylating ULK1

Unc-51-like autophagy activating kinase 1 (ULK1), a mammalian homolog of the yeast kinase Atg1, has an essential role in autophagy induction. In nutrient and growth factor signaling, ULK1 activity is regulated by various posttranslational modifications, including phosphorylation, acetylation, and ubiquitination. We previously identified glycogen synthase kinase 3 beta (GSK3B) as an upstream regulator of insulin withdrawal-induced autophagy in adult hippocampal neural stem cells. Here, we report that following insulin withdrawal, GSK3B directly interacted with and activated ULK1 via phosphorylation of S405 and S415 within the GABARAP-interacting region. Phosphorylation of these residues facilitated the interaction of ULK1 with MAP1LC3B and GABARAPL1, while phosphorylation-defective mutants of ULK1 failed to do so and could not induce autophagy flux. Furthermore, high phosphorylation levels of ULK1 at S405 and S415 were observed in human pancreatic cancer cell lines, all of which are known to exhibit high levels of autophagy. Our results reveal the importance of GSK3B-mediated phosphorylation for ULK1 regulation and autophagy induction and potentially for tumorigenesis. © 2021, The Author(s).1

ATG101 Degradation by HUWE1-Mediated Ubiquitination Impairs Autophagy and Reduces Survival in Cancer Cells

Autophagy is a critical cytoprotective mechanism against stress, which is initiated by the protein kinase Unc-51-like kinase 1 (ULK1) complex. Autophagy plays a role in both inhibiting the progression of diseases and facilitating pathogenesis, so it is critical to elucidate the mechanisms regulating individual components of the autophagy machinery under various conditions. Here, we examined whether ULK1 complex component autophagy-related protein 101 (ATG101) is downregulated via ubiquitination, and whether this in turn suppresses autophagy activity in cancer cells. Knockout of ATG101 in cancer cells using CRISPR resulted in severe growth retardation and lower survival under nutrient starvation. Transfection of mutant ATG101 revealed that the C-terminal region is a key domain of ubiquitination, while co-immunoprecipitation and knockdown experiments revealed that HECT, UBA and WWE domain containing E3 ubiquitin protein ligase 1(HUWE1) is a major E3 ubiquitin ligase targeting ATG101. Protein levels of ATG101 was more stable and the related-autophagy activity was higher in HUWE1-depleted cancer cells compared to wild type (WT) controls, indicating that HUWE1-mediated ubiquitination promotes ATG101 degradation. Moreover, enhanced autophagy in HUWE1-depleted cancer cells was reversed by siRNA-mediated ATG101 knockdown. Stable ATG101 level in HUWE1-depleted cells was a strong driver of autophagosome formation similar to upregulation of the known HUWE1 substrate WD repeat domain, phosphoinositide interacting 2 (WIPI2). Cellular survival rates were higher in HUWE1-knockdown cancer cells compared to controls, while concomitant siRNA-mediated ATG101 knockdown tends to increase apoptosis rate. Collectively, these results suggest that HUWE1 normally serves to suppress autophagy by ubiquitinating and triggering degradation of ATG101 and WIPI2, which in turn represses the survival of cancer cells. Accordingly, ATG101-mediated autophagy may play a critical role in overcoming metabolic stress, thereby contributing to the growth, survival, and treatment resistance of certain cancers

Changes in Human Electroencephalographic Activity in Response to <i>Agastache rugosa</i> Essential Oil Exposure

Agastache rugosa (Korean mint) is an important medicinal and aromatic plant and its aerial parts have a pleasant fragrance. A. rugosa leaves are used as an ingredient in salads and soups for enhancing the aroma and taste of foods in Korea. However, there is no report on the influence of the aroma of A. rugosa on human psychophysiological activity. Therefore, the present study aimed to investigate the effect of exposure to the essential oil of Korean A. rugosa on human electroencephalographic (EEG) activity. The essential oil of A. rugosa was isolated using steam distillation extraction and its composition was determined by gas chromatography and mass spectrometry (GC–MS) analysis. In the EEG study, 38 healthy volunteers (19 men and 19 women) participated. The EEG readings were analyzed for 25 EEG indices from 29 electrodes placed on the scalp according to the international 10–20 system. The major component in the essential oil of A. rugosa was estragole (89.49%) followed by D-limonene (3.40%), menthone (1.80%), and pulegone (1.86%). In the EEG study, significant decreases in absolute theta (AT) and relative theta (RT) power spectra were observed during the exposure to A. rugosa essential oil when compared to that of no odor exposure. Whereas relative alpha (RA), relative slow alpha (RSA), spectral edge frequency 50% (SEF50), and spectral edge frequency 50% of alpha (ASEF) power spectra values significantly increased. These results reveal that the EEG power spectra changes incurred during the exposure to the essential oil of A. rugosa may be associated with the enhancement of freshness and concentration states of the human brain

Direct Interaction of CD40 on Tumor Cells with CD40L on T Cells Increases the Proliferation of Tumor Cells by Enhancing TGF-β Production and Th17 Differentiation.

It has recently been reported that the CD40-CD40 ligand (CD40L) interaction is important in Th17 development. In addition, transforming growth factor-beta (TGF-β) promotes tumorigenesis as an immunosuppressive cytokine and is crucial in the development of Th17 cells. This study investigated the role of CD40 in breast cancer cells and its role in immunosuppressive function and tumor progression. CD40 was highly expressed in the breast cancer cell line MDA-MB231, and its stimulation with CD40 antibodies caused the up-regulation of TGF-β. Direct CD40-CD40L interaction between MDA-MB231 cells and activated T cells also increased TGF-β production and induced the production of IL-17, which accelerated the proliferation of MDA-MB231 cells through the activation of STAT3. Taken together, the direct CD40-CD40L interaction of breast tumor cells and activated T cells increases TGF-β production and the differentiation of Th17 cells, which promotes the proliferation of breast cancer cells

CD40 ligand stimulation on the activated T cells induces Th17 differentiation.

<p>(A) Human T cells (2.5x10⁶/ml) purified from human PBMCs were activated as described in <i>Materials and Methods</i>. After increase of CD40L expression on activated T cells was confirmed by flow cytometry analysis, they were co-cultured with MDA-MB231 cells in the same well on 6-well plate at the ratio of 5:1 for 24 hrs. Th17 differentiation was confirmed by staining of intracellular IL-17 in CD4⁺ T cells with Alexa Fluor 647-conjugated anti-human IL-17A antibody (1 μg/ml) and FITC-conjugated anti-human CD4 antibody (1 μg/ml). To confirm the role of CD40 on Th17 differentiation, the interaction between CD40 and CD40L was interfered with the addition of anti-CD40 neutralizing antibody (2 μg/ml) for 1 hr. Result is the representative of three independent experiments. (B-D) Culture supernatant from the experiment described in Fig 3A were collected and the amount of IL-1β (B), IL-6 (C) and IL-21 (D) were measured by ELISA, according to manufacturer’s instruction. Data represents mean ± S.D. Result is the representative of three independent experiments and each experiment was performed in triplicate. *p < 0.05. (E) Human T cells (2.5x10⁶/ml) purified from human PBMCs were activated as described in <i>Materials and Methods</i>. After increase of CD40L expression on activated T cells was confirmed by flow cytometry analysis, activated T cells were stimulated by anti-CD40L agonistic antibody (1 μg/ml) or isotype (1 μg/ml) for 24 and 36 hrs. The expression of ROR gamma t (RORγt), the orphan nuclear receptor for IL-17 transcription and Th17 differentiation was determined by intracellular flow cytometry analysis, after staining with PE-conjugated anti- RORγt antibody (1 μg/ml) and PE-conjugated Rat IgG (1 μg/ml) as described in <i>Materials and Methods</i>. Result is the representative of three independent experiments.</p

IL-17 production via direct interaction of CD40 and CD40L increases STAT3 activation and the proliferation of MDA-MD231 cells.

<p>(A) MDA-MB231 cells and activated T cells were directly co-cultured at the ratio of 1:5 for 24 hrs in the presence of anti-IL-17 neutralizing antibody or anti-CD40 neutralizing antibody (2 μg/ml/each) on 96-well plate, and then cells were cultured for 24 hrs. After the addition of 1 μCi/mL of [³H]-thymidine, cells were culture for another 18 hrs. And the proliferation of cells was measured as described in <i>Materials and Methods</i>. Data represents mean ± S.D. The assay was performed in quadruplicate and result is the representative of three independent experiments. (B) MDA-MB231 cells were cultured in the presence of recombinant IL-17 (rIL-17, 50 ng/ml) for 15, 30 and 60 min. In addition, cells were cultured with direct co-culture supernatant of MDA-MB231 cells and activated T cells in the presence or absence of anti-IL-17 neutralizing antibody (nAb). And then, the activation of STAT3 was examined by western blot as described in <i>Materials and Methods</i>. Lane 1: Control, Lane 2: rIL-17 (50 ng/ml), Lane 3: Direct co-culture supernatant of MDA-MB231 cells and activated T cells, Lane 4: Direct co-culture supernatant of MDA-MB231 cells and activated T cells with IL-17 nAb, Lane 5: Indirect co-culture supernatant of MDA-MB231 cells and activated T cells. β-actin was used as a loading control. Result is the representative of three independent experiments. (C) MDA-MB231 cells and activated T cells were directly co-cultured at the ratio of 1:5 for 24 hrs in the presence of 20 μM of AG490 (STAT3 inhibitor) or anti-IL-17 nAb (2 μg/ml) on 96-well plate, and then cells were cultured for 24 hrs. After the addition of 1 μCi/mL of [³H]-thymidine, cells were culture for another 18 hrs. Direct co-culture supernatant of CD40-non expressing breast cancer cell line, Hs578T and activated T cells was used as a negative control. The proliferation of cells was measured as described in <i>Materials and Methods</i>. Data represents mean ± S.D. The assay was performed in quadruplicate and result is the representative of three independent experiments.</p

TGF-β production is up-regulated by CD40 stimulation in a breast cancer cell line, MDA-MB231.

<p>(A) The breast cancer cell lines, MDA-MB231 and Hs578T were collected at continuous log phase of growth. The expression of CD40 was examined by staining with PE-conjugated anti-human CD40 antibody (1 μg/ml) as described in <i>Materials and Methods</i>. Result is the representative of three independent experiments. (B) MDA-MB231 cells (2.5 x 10³/well) were stimulated with the various concentrations of anti-CD40 agonistic antibody (2, 4, 8, and 16 μg/ml) and the same amount of isotype antibody for 1 hr on 96-well plate, and then cells were cultured for 24 hrs. After the addition of 1 μCi/mL of [³H]-thymidine, cells were culture for another 18 hrs. And then, the proliferation of cells by CD40 stimulation was measured as described in <i>Materials and Methods</i>. Data represents mean ± S.D. The assay was performed in quadruplicate and result is the representative of three independent experiments. There was no statistical significance among groups. (C and D) The expression of TGF-β mRNA transcript in MDA-MB231 cells by CD40 stimulation was investigated with semi-quantitative RT-PCR (C) and quantitative real-time PCR (D). Cells (1.5 x 10⁵/well) were stimulated 2 μg/ml of anti-CD40 agonistic antibody for 3, 6, 9 and 12 hrs on 6-well plate. Cells were harvested and total RNA was extracted with Trizol, according to manufacturer’s instruction. After quantification, 1μg of RNA was converted to cDNA by reverse transcriptase. And then RT-PCR and real-time PCR were performed with specific primers for TGF-β as described in <i>Materials and Methods</i>. β-actin or GAPDH was used as a loading control. ***p < 0.001. (E) CD40 on MDA-MB231 cells (1.5 x 10⁵/well) were stimulated with anti-CD40 agonistic antibody (2 and 4 μg/ml) and soluble CD40L (3 μg/ml) for 1 hr, and then cells were cultured for 24 hrs on 6-well plate. The amount of TGF-β in the culture supernatant was measured by ELISA, according to manufacturer’s instruction. Data represents mean ± S.D. Result is the representative of three independent experiments and each experiment was performed in triplicate. **p < 0.01 vs. untreated control. (F) Cells (1.5 x 10⁵/well) were seeded on 6-well plate and then transfected with 20 nM of CD40 siRNA and control siRNA in the mixture of serum free media and oligofectamine as described in <i>Materials and Methods</i>. After 72 hrs, the down-regulation of CD40 expression on MDA-MB231 cells by CD40 siRNA transfection was confirmed by flow cytometry analysis. And then, cells (1.5 x 10⁵/well) were stimulated with anti-CD40 agonistic antibody (2 μg/ml) and soluble CD40L (3 μg/ml) for 1 hr, and then cells were cultured for 24 hrs on 6-well plate. And then, the amount of TGF-β in the culture supernatant was measured by ELISA. Data represents mean ± S.D. Result is the representative of three independent experiments and each experiment was performed in triplicate. ***p < 0.001 vs. untreated control.</p