Neural stem cell-conditioned medium upregulated the PCMT1 expression and inhibited the phosphorylation of MST1 in SH-SY5Y cells induced by Aβ_25-35

A progressive neurodegenerative disease, Alzheimer’s disease (AD). Studies suggest that highly expressed protein isoaspartate methyltransferase 1 (PCMT1) in brain tissue. In the current study, we explored the effects of neural stem cell-conditioned medium (NSC-CDM) on the PCMT1/MST1 pathway to alleviate Aβ_25-35-induced damage in SH-SY5Y cells. Our data suggested that Aβ25-35 markedly inhibited cell viability. NSC-CDM or Neural stem cell-complete medium (NSC-CPM) had a suppression effect on toxicity when treatment with Aβ_25-35, with a greater effect observed with NSC-CDM. Aβ_25-35 + NSC-CDM group exhibited an increase in PCMT1 expression. sh-PCMT1 markedly decreased cell proliferation and suppressed the protective role of NSC-CDM through the induction of apoptosis and improved p-MST1 expression. Overexpression of PCMT1 reversed the Aβ_25-35-induced decrease in cell proliferation and apoptosis. In summary, our findings suggest that NSC-CDM corrects the Aβ_25-35- induced damage to cells by improving PCMT1 expressions, which in turn reduces phosphorylation of MST1.     

Proteomics analysis provides novel biomarkers and therapeutic target candidates in the treatment of the Huang-Pu-Tong-Qiao formula in an AD rat model

Background: Huang-Pu-Tong-Qiao formula (HPTQ), a traditional Chinese herbal formula, has a variety of pharmacological effects. It has been used to treat Alzheimer’s disease (AD) for decades. This study aimed to screen differentially expressed proteins in the hippocampus of AD model rats treated with HPTQ. Proteomic studies of the effects of HPTQ on AD are key to understanding the therapeutic mechanisms of HPTQ and identifying potential therapeutic targets. Methods: We hence used the isobaric tags for relative and absolute quantification (ITRAQ) approach to investigate the differentially expressed proteins in the hippocampus of AD model rats before and after HPTQ administration and to identify the potential therapeutic target proteins of HPTQ. In this study, the learning and memory abilities of AD rats were examined by the Morris water maze test. After HPTQ administration, the differentially expressed proteins in the hippocampus of AD rats were quantified and analyzed in silico. Furthermore, western blotting was used to verify the expression of related proteins. Results: The Morris water maze results showed that HPTQ could improve the learning and memory ability of AD model rats. The proteomics analysis results showed that 57 proteins were differentially expressed, of which 35 were up-regulated and 22 were down-regulated. Bioinformatics analysis indicated that proteins with altered expression after HPTQ treatment were involved in several biological processes that have the potential to exert neuroprotective effects. These included promoting the translation of ribosomes, improving the deposition of amyloid-beta (Aβ), regulating autophagy, regulating neuronal synaptic function and plasticity, and alleviating oxidative stress. Conclusion: In conclusion, we identified several potential therapeutic target proteins and related mechanistic pathways of HPTQ in the treatment of AD, laying the foundation for further investigation of the therapeutic effects of HPTQ.     

Mesenchymal stem cells: As a multi-target cell therapy for clearing β-amyloid deposition in Alzheimer’s disease

Extracellular β-amyloid (Aβ) plaques and neurofibrillary tangles (NFTs) are the pathological hallmarks of Alzheimer’s disease (AD). Studies have shown that aggregates of extracellular Aβ can induce neuroinflammation mediated neurotoxic signaling through microglial activation and release of pro-inflammatory factors. Thus, modulation of Aβ might be a potential therapeutic strategy for modifying disease progression. Recently, a large number of reports have confirmed the beneficial effects of mesenchymal stem cells (MSCs) on AD. It is believed to reduce neuroinflammation, reduce Aβ amyloid deposits and NFTs, increase acetylcholine levels, promote neurogenesis, reduce neuronal damage, and improve working memory and cognition. In this review, we focus on the role of MSCs in clearing Aβ deposition. MSCs have the potential to modulate Aβ-related microenvironments via enhancement of autophagy, proteolysis of Aβ aggregates, phagocytic clearance of Aβ by microglial M2 polarization, decrease oxidative stress (OS), and correction of abnormal sphingolipid (SL) metabolism. With advantages in clinical applications, these data suggest that the use of MSCs as a multi-target modulator of Aβ would be an effective therapeutic approach in AD.     

AKT regulates IL-1β-induced proliferation and activation of hepatic stellate cells

Background: Activated hepatic stellate cells (HSCs) are closely involved in the initiation, perpetuation, and resolution of liver fibrosis. Pro-inflammatory cytokine levels are positively correlated with the transition from liver injury to fibrogenesis and contribute to HSC pathophysiology in liver fibrosis. Methods: In this study, we investigated the effect of the pro-inflammatory cytokine interleukin (IL)-1β on the proliferation and signaling pathways involved in fibrogenesis in LX-2 cells, an HSC cell line, using western blotting and cell proliferation assays. Results: IL-1β increased the proliferation rate and α-smooth muscle actin (SMA) expression of LX-2 cells in a dose-dependent manner. Within 1 h after IL-1β treatment, c-Jun N-terminal kinase (JNK), p38, and nuclear factor-κB (NF-κB) signaling was activated in LX-2 cells. Subsequently, protein kinase B (AKT) phosphorylation and an increase in α- SMA expression were observed in LX-2 cells. Each inhibitor of JNK, p38, or NF-κB decreased cell proliferation, AKT phosphorylation, and α-SMA expression in IL-1β-treated LX-2 cells. Conclusion: These results indicate that JNK, p38, and NF-κB signals converge at AKT phosphorylation, leading to LX-2 activation by IL-1β. Therefore, the AKT signaling pathway can be used as a target for alleviating liver fibrosis by the inflammatory cytokine IL-1β.     

LncRNA <i>ZFAS1</i> regulates cardiomyocyte differentiation of human embryonic stem cells

Background: Cardiomyocytes derived from human embryonic stem cells (hESCs) are regulated by complex and stringent gene networks during differentiation. Long non-coding RNAs (lncRNAs) exert critical epigenetic regulatory functions in multiple differentiation processes. However, the involvement of lncRNAs in the differentiation of hESCs into cardiomyocytes has not yet been fully elucidated. Here, we identified the key roles of ZFAS1 (lncRNA zinc finger antisense 1) in the differentiation of cardiomyocytes from hESCs. Methods: A model of cardiomyocyte differentiation from stem cells was established using the monolayer differentiation method, and the number of beating hESCs-derived cardiomyocytes was calculated. Gene expression was analyzed by quantitative real-time PCR (qRT-PCR). Immunofluorescence assays were performed to assess the expression of cardiac troponin T (cTnT) and α-actinin protein in cardiomyocytes. Results: qRT-PCR showed that ZFAS1 expression in the mesoderm was significantly higher than that in embryonic stem cells, cardiac progenitor cells, and cardiomyocytes. Knockdown of ZFAS1 inhibited cardiomyocyte differentiation from hESCs, which was characterized by reduced expression of the cardiac-specific markers cTnT, α-actinin, myosin heavy chain 6 (MYH6), and myosin heavy chain 7 (MYH7). In contrast, ZFAS1 overexpression remarkably increased the percentage of spontaneously beating cardiomyocytes. In terms of the mechanism, we found that ZFAS1 is an antisense lncRNA at the 5′ end of the protein-coding gene ZNFX1. Knockdown of ZFAS1 could increase the mRNA expression level of ZNFX1. Furthermore, qRT-PCR demonstrated that the silencing of ZNFX1 led to an increase in cardiac-specific markers that predicted the promotion of cardiomyocyte differentiation. Conclusion: Altogether, these data suggest that lncRNA-ZFAS1 is required for cardiac differentiation by functionally inhibiting the expression of ZNFX1, which may provide a reference for the treatment of heart disease to a certain extent.     

The neuroprotection of electro-acupuncture via the PGC-1α/TFAM pathway in transient focal cerebral ischemia rats

ATP depletion is one of the pathological bases in cerebral ischemia. Electro-acupuncture (EA) is widely used in clinical practice for ischemia. However, the mechanism of EA remains unclear. The purpose of this study was to investigate whether EA could activate the AMPK/PGC-1α/TFAM signaling pathway and, consequently, increase the preservation of ATP in rats with ischemia. In this study, 48 rats were randomly divided into four groups as a sham-operation control group (sham group), a middle cerebral artery occlusion group (MCAO group), an EA group, and an EA group blocked by the AMPK inhibitor compound C (EA + CC group) (N = 12/group). The rats of the EA group and EA + CC group received the EA treatment for 7 days. The rats that belonged in the two remaining groups were only grasped in the same condition. Then, their brain tissues were collected for further detection. When compared with other groups, EA significantly reduced neurological deficits score and increased motor function. The cerebral infarction volume was significantly reduced in the EA group according to TTC staining. With western blot, we found that EA improved the ratio of p-AMPKα/AMPKα (P < 0.05), however, there is no difference between the MCAO group and sham group (P > 0.05). In addition, EA also increased the expression of PGC-1α and TFAM (all P < 0.05). By Elisa, we observed that EA increased the preservation of ATP (P < 0.05) and mitochondrial respiratory enzymes, including Complex I (P < 0.05), Complex IV (P < 0.05), but not Complex III (P > 0.05). In summary, we conclude that EA may protect against ischemic damage in MCAO rats, improve the preservation of ATP and mitochondrial respiratory enzymes. This effect may be positively regulated by the activation of the PGC-1α/TFAM signaling pathway.     

Cardioprotective effect of ivabradine via the AMPK/SIRT1/PGC-1α signaling pathway in myocardial ischemia/reperfusion injury induced in H9c2 cell

Post-resuscitation myocardial dysfunction (PRMD) is the most severe myocardial ischemia-reperfusion injury (MIRI) and is characterized by difficult treatment and poor prognosis. Research has shown the protective effects of the rational use of ivabradine (IVA) against PRMD; however, the molecular mechanisms of IVA remain unknown. In this study, an ischemia-reperfusion injury (IRI) model was established using hypoxic chambers. The results demonstrated that pretreatment with IVA reduced IRI-induced cytotoxicity and apoptosis. IVA attenuated mitochondrial damage, eliminated excess reactive oxygen species (ROS), suppressed IRI-induced ATP and NAD⁺ , and increased the AMP/ATP ratio. We further found that IVA increased the mRNA levels of sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and upregulated the expression levels of phosphorylated AMP-activated protein kinase (p-AMPK)/AMPK, SIRT1, and PGC-1α proteins. Interestingly, no change in AMPK mRNA levels was observed. Cardiomyocyte energy metabolism significantly changed after IRI. The aim of this study was to demonstrate the cardioprotective effect of Ivabradine via the AMPK/SIRT1/PGC-1α signaling pathway in myocardial ischemia/reperfusion injury-induced in H9c2 cell.     

Capsaicin exerts anti-benign prostatic hyperplasia effects via inhibiting androgen receptor signaling pathway

Background: Benign prostatic hyperplasia (BPH) is a common condition in middle-aged and elderly men. Enlargement of the prostate causes lower urinary tract symptoms. Capsaicin is a phytochemical extracted from chili peppers and exerts many pharmacological actions, such as anti-tumor and anti-inflammatory effects. Methods: Our study investigated the effect of capsaicin in vitro and in a mouse model in vivo. A prostatic stromal myofibroblast cell line (WPMY-1) was co-incubated with testosterone (1 µM) and different concentrations of capsaicin (10–100 µM) for 24 and 48 h. Capsaicin (10–100 µM) significantly inhibited testosterone-treated WPMY-1 cell growth at 48 h by MTT assay. The testosterone propionate (7.5 mg/kg)-induced BPH mouse model was used to examine the anti-proliferative effect of capsaicin. Treatment with capsaicin (10 mg/kg) for 14 days significantly attenuated prostatic hyperplasia. Finasteride was used as a positive control. Results: Capsaicin significantly decreased prostate weight and prostate index (prostate/body weight ratio) in BPH mice. The expression of 5α-reductase type II, androgen receptor (AR) and prostate specific antigen (PSA) protein expression and PSA serum were all significantly reduced in capsaicin-treated BPH mice. In addition, capsaicin also activated transient receptor potential vanilloid 1 mediated apoptosis and autophagy in BPH mice. Conclusion: These results demonstrate multiple positive effects of capsaicin in controlling prostate growth and suggest its therapeutic potential in the treatment of BPH.     

Effect of Peroxiredoxin 1 on the biological function of airway epithelial cells and epithelial-mesenchymal transition

Peroxiredoxin 1 (PRDX1) participates in tumor cell proliferation, apoptosis, migration, invasion, and the epithelial-to-mesenchymal transition (EMT). This study aimed to investigate the effect of PRDX1 on the EMT of airway epithelial cells stimulated with lipopolysaccharide (LPS) and transforming growth factor-beta 1 (TGF-β1). PRDX1 overexpression significantly increased the proliferation and migration of human bronchial epithelial (BEAS-2B) cells, reduced cell apoptosis (p < 0.01), and induced EMT and collagen deposition by upregulating the expression of the matrix metallopeptidase (MMP)2, MMP9, α-smooth muscle actin (α-SMA), N-cadherin, vimentin and twist proteins and inhibiting E-cadherin expression (p < 0.05). PRDX1 overexpression promoted TGF-β1-mediated inhibition of cell proliferation and migration and significantly enhanced the TGF-β1-induced EMT and collagen synthesis (p < 0.05). Knockdown of PRDX1 inhibited cell proliferation, migration, EMT, and collagen synthesis (p < 0.01), reversed LPS-mediated inhibition of cell proliferation and migration, and significantly suppressed LPS-induced EMT and collagen synthesis (p < 0.01). The result indicating that PRDX1 may be involved in LPS/TGF-1-induced EMT and collagen synthesis in human bronchial epithelial cells.     

Hypoxia induced apoptosis of rat gastric mucosal cells by activating autophagy through HIF-1α/TERT/mTORC1 pathway

The pathogenesis of high altitude-related gastric mucosal injury remains poorly understood, this study aimed to investigate the role of autophagy in hypoxia-induced apoptosis of rat gastric mucosal cells. Rats were randomized into four groups which were maintained at an altitude of 400 m (P) or received no treatment (H), autophagy inducer rapamycin (H+AI) or autophagy inhibitor 3-MA (H+AB) at an altitude of 4,300 m for 1, 7, 14 and 21 days, respectively, and the morphology, ultrastructure, autophagy, and apoptosis of gastric mucosal tissues were examined. Gastric mucosal epithelial cells CC-R039 were cultured under conditions of normoxia, 2% O2 (hypoxia), or 2% O2+anti-mTORC1 for 0, 24, 48, and 72 h, respectively, and the autophagy and apoptosis were analyzed. CC-R039 cells were transfected with siHIF-1α, siTERT, or siRNA and the autophagy was examined. The results showed that the exposure to hypoxia increased the autophagy and apoptosis of gastric mucosal cells in rats, and apoptosis was aggravated by rapamycin treatment but alleviated by 3-MA treatment. Increased duration of hypoxia from 0 to 72 h could increase the autophagy and apoptosis but decrease the proliferation of gastric mucosal cells. Inhibition of mTORC1 with rapamycin led to further increase in apoptosis and even substantial cell death, and inhibition of HIF- 1α and TERT increased mTORC1 expression and reduced autophagy. Moreover, the inhibition of HIF-1α reduced TERT expression. In conclusion, hypoxia could induce apoptosis of rat gastric mucosal cells by activating autophagy through HIF-1α/TERT/mTORC1 pathway     

Tanshinone IIA protects intestinal epithelial cells from ferroptosis through the upregulation of <i>GPX4</i> and <i>SLC7A11</i>

Background: Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract. The destruction of the intestinal epithelial barrier is one of the major pathological processes in IBD pathology. Growing evidence indicated that epithelial cell ferroptosis is linked to IBD and is considered a target process. Methods: RAS-selective lethal 3 (RSL3) was used to induce ferroptosis in intestinal epithelial cell line No. 6 (IEC-6) cells, and cell ferroptosis and the effects of tanshinone IIA (Tan IIA) were determined by cell counting kit-8 (CCK-8), reactive oxygen species (ROS) staining, Giemsa staining and transmission electron microscope (TEM). The cell viability of natural product library compounds was determined by CCK-8. The expression of ferroptosis-related genes were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. Results: Treatment of IEC-6 cells results in the accumulation of ROS and typical morphological characteristics of ferroptosis. RSL3 treatment caused rapid cellular cytotoxicity which could be reversed by ferrostatin-1 (Fer-1) in IEC-6 cells. Natural product library screening revealed that Tan IIA is a potent inhibitor of IEC-6 cell ferroptosis. Tan IIA could significantly protect the RSL3-induced ferroptosis of IEC-6 cells. Furthermore, the ferroptosis suppressors, glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), and miR-17-92 were found to be early response genes in RSL3-treated cells. Treatment of IEC-6 cells with Tan IIA resulted in upregulation of GPX4, SLC7A11, and miR-17-92. Conclusion: Our study demonstrated that Tan IIA protects IEC-6 cells from ferroptosis through the upregulation of GPX4, SLC7A11, and miR-17-92. The findings might provide a theoretical grounding for the future application of Tan IIA to treat or prevent IBD.     

Overexpression of inhibin α (1-32) fusion protein promotes apoptosis and cell cycle arrest in a cervical cancer cell model (Hela cells)

Inhibins play important roles in the reproductive system. To evaluate whether inhibin α (1-32) fusion protein plays a role in cervical cancer growth, the plasmid pVAX-inhα was constructed and its effect on proliferation and apoptosis of the human cervical cancer cell line (Hela) was checked by flow cytometry and real-time PCR. The expression and localization of inhibin α protein were detected by RT-PCR and confocal microscopy which showed that inhibin α protein was expressed and localized in the nucleus of Hela cells. Over expression of inhibin α gene significantly induced cell apoptosis and ceased S phase of cell cycle. Furthermore, cell proliferation was significantly suppressed 96 h post-transfection and mRNA level of anti-apoptosis genes (Bcl-2, NFκB) were decreased but pro-apoptosis genes (Bax, wild type p53) and inhibin co receptor (TGFβR3) were increased, indicating that inhibin, through its co-receptor, might activate apoptotic and cell growth cascades which regulate proliferation and apoptosis in Hela cells. These results suggest that inhibin α (1-32) fusion protein, located in the cell nucleus, can regulate Hela cells growth and apoptosis by induction of apoptotic pathways such as NFκB, Bcl-2 and p53 families. These findings may have a significant impact on future research regarding cervical cancer cell lines     

Organogenesis and plant regeneration of <i>Arachis villosa</i> Benth. (Leguminosae) through leaf culture

With the aim of developing an efficient plant regeneration protocol, leaflet explants of three accessions of Arachis villosa Benth. (S2866, S2867 and L97) were cultured on basic Murashige and Skoog medium supplemented with different combinations of plant growth regulators: α-naphthalenacetic acid, indole-3-butyric acid, 6-benzylaminopurine, kinetin and thidiazuron. The accession L97 was the only one able to differentiate buds through indirect organogenesis. The most suitable combination for bud regeneration was the basic medium added with 13.62 μM thidiazuron and 4.44 μM 6-benzylaminopurine. These results show the important role of the genotype in morphogenetic responses and the organogenetic effect of thidiazuron in Arachis villosa accession L97. A thidiazuron lacking media (only 0.54 μM α-naphthalenacetic acid, 13.95 μM kinetin and 13.32 μM 6-benzylaminopurine were added) promoted the elongation of the regenerated buds. Adventitious rooting was achieved 90 days after the isolated shoots were transferred to a rooting medium containing 0.54 μM α-naphthalenacetic acid.     

Association of hypoxia-inducible factor-1α (HIF1α) 1772C/T gene polymorphism with susceptibility to renal cell carcinoma/prostate cancer

In this study, we used a meta-analysis method to evaluate the relationship between hypoxia-inducible factor-1α (HIF1α) 1772C/T gene polymorphism (rs 11549465) and renal cell carcinoma (RCC)/prostate cancer risk. We searched for relevant studies (before March 1, 2019) on Cochrane Library, Embase, and PubMed. Studies meeting the inclusion criteria were recruited into this meta-analysis. The outcome of dichotomous data was showed in the way of odds ratios (OR), and 95% confidence intervals (CI) were also counted. In this investigation, there was no association between HIF1α 1772C/T gene polymorphism and susceptibility to RCC in Caucasians, Asians as well as overall populations. In addition, HIF1α 1772C/T gene polymorphism was not found to be relevant to the survival in RCC. Interestingly, the T allele was relevant to prostate cancer risk in all populations, but not in Caucasians and Asians. However, the TT genotype and the CC genotype were not related to prostate cancer susceptibility in Asian, Caucasian, and all populations. In conclusion, the T allele of the HIF1α 1772C/T gene polymorphism was related to prostate cancer risk in the overall populations.     

<i>LINC00609</i> inhibits A549 cells progression through the regulation of <i>miR-128-3p/RND3</i> axis

Background: Long-chain non-coding RNA (lncRNA) LINC00609 is a potential tumor suppressor, but the mechanism of action in non-small cell lung cancer (NSCLC) is yet to be understood.Objectives: The effects of LINC00609 on A549 cell proliferation, apoptosis, and cell cycle arrest were investigated. Methods: The LINC00609 levels in NSCLC and normal tissues were analyzed by bioinformatics. Expressions of LINC00609, miR-128-3p, and Rho family GTPase 3 (RND3) in NSCLC cells (A549) were determined by qRT-PCR. Bioinformatics analysis predicted target genes and dual-luciferase reporter assays to ensure that LINC00609 targeted miR-128-3p and miR-128-3p targeted RND3. The proliferation of cells was determined using EDU and CCK-8. Flow cytometry was used to evaluate cell apoptosis rate and cell cycle. The western blotting assay identified proteins related to proliferation and apoptosis. Results: In NSCLC tissues, LINC00609 was expressed in low levels, while its high expression was associated with a higher survival rate. LINC00609 affected cell proliferation, apoptosis, cell cycle arrest, and expression of related proteins. Dual-luciferase reporter assay showed that LINC00609 binds specifically to miR-128-3p, and miR-128-3p binds to RND3. MiR-128-3p overexpression could neutralize the effects of LINC00609. A siRNA targeting RND3 could reverse the effect of the miR-128-3p inhibitor. Silencing RND3 resulted in a decrease in apoptosis rate and the number of cells in the S-phase and an increase in the number of cells in the G1-phase. Furthermore, phosphorylation levels of the AKT protein and mTOR protein, and Bcl2 expression, increased; however, the expression of RND3, Bax, and caspase3 decreased. Conclusions: LINC00609 regulated miR-128-3p/RND3 axis to modulate A549 cell proliferation, apoptosis, and cell cycle arrest. In the case of NSCLC, LINC00609 could be a potential target for therapy.     

Oxidative effects of glyphosate on the lipophobic intracellular environment in the microalgae <i>Chlorella vulgaris</i>

The studied hypothesis is that the herbicide glyphosate (GLY) can affect the oxidative balance in the hydrophobic intracellular medium in non-target Chlorella vulgaris cells. Analytical GLY and RoundUp (RUP) supplementation, affected the growth profile. A significant 42% decrease in the cellular biomass in stationary (St) phase was observed in cultures supplemented with either 5 µM of GLY or RUP, as compared to control cultures. The treatment with 0.3 µM of GLY generated non-significant effects on the oxidation rate of 2’, 7’ dichlorofluorescein diacetate (DCFH-DA), neither in exponential (Exp) nor in St phase of development, as compared to control cultures. However, the treatment with either 5 µM GLY or 0.3 and 5 µM RUP lead to a significant decrease in the DCFH-DA oxidation rate, as compared to control cultures. The lipid radical (LR^●) generation rate, detected by Paramagnetic Resonance Spectroscopy (EPR), was significantly increased in the presence of RUP, in Lag and Exp phase of growth. The non-enzymatic antioxidants, α-Tocopherol (α-T) and β-Carotene (β-C), are aimed to protect membranes against the damage produced by the radical reactions. The content of β-C was not significantly affected, as compared to control cultures, by any of the treatments, in both growth phases of cellular development. The content of α-T was significantly decreased by the supplementation with either 0.3 or 5 µM of RUP or 5 µM GLY. The LR^●/α-T ratio, used as indicator of the oxidative balance in the hydrophobic cellular media, was significantly different between samples obtained from control and RUP-exposed microalgae in both, Exp and St phase of development, with either 0.3 or 5 μM RUP. The data presented here showed evidence that suggested that oxidative balance in the hydrophobic environment was affected by either GLY or RUP.