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Original Research ARTICLE
Glycine-Histidine-Lysine (GHK) Alleviates Astrocytes Injury of Intracerebral Hemorrhage via the Akt/miR-146a-3p/AQP4 Pathway
- 1Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
- 2Guangdong Provincial Key Laboratory of Diagnosis and Treatment of Major Neurological Diseases, National Key Clinical Department and Key Discipline of Neurology, Guangzhou, China
- 3Department of Neurology, First Hospital of China Medical University, Shenyang, China
Intracerebral hemorrhage (ICH) is a major type of cerebrovascular disease with poor prognosis. Recent studies have shown that Glycyl-l-histidyl-l-lysine (GHK) is a kind of natural human tripeptide which could inhibit inflammation and against neurodegenerative diseases, but neither its role nor the mechanisms in ICH have yet been explicit. Currently, we investigated the possible strategies of GHK on ICH injury. Neurological deficit scores, brain water content, Nissl staining, and aquaporin 4 (AQP4) immunohistochemistry were detected in different groups of rats. The expression of microRNAs (miRNAs) was examined by real-time PCR. Inflammatory factors were detected using enzyme-linked immunosorbent assay (ELISA). Cell viability and cell proliferation were detected by Cell Counting Kit-8 (CCK-8). Matrix metalloproteinase 2 (MMP2), MMP9, tissue inhibitors of metalloproteinase-1 (TIMP1), AQP4 expression were detected/assessed using western blot. We observed that 5 and 10 μg/g of GHK improved neurological recovery by significantly reducing brain water content, improving neurological deficits, and promoting neuron survival. Besides, GHK alleviated inflammatory reaction and downregulated AQP4 expression. Furthermore, the effects of GHK on astrocyte were associated with the upregulation of miRNA-146a-3p, which partially regulated the expression of AQP4. Our results demonstrated that the phosphatidylinositol 3-kinase (PI3K)/AKT pathway participated in the GHK-induced upregulation of miR-146a-3p and miR-146a-3p/AQP4 interaction plays a role in the injury following ICH. These findings suggested that GHK could provide a novel therapeutic strategy for ICH.
Stroke is a leading cause of disability and mortality globally. Intracerebral hemorrhage (ICH) accounts for 15–20% of all stroke incidents, with the highest mortality in all subtypes of stroke, leading to serious social burden (Huang et al., 2019; Lo and Teitelbaum, 2020). In ICH, the initial injury by the rupture of vessels and the compression of the expanding hematoma is mostly followed by secondary damage, which involves hemoglobin cytotoxicity, microglial activation, and inflammatory cell infiltration in the perihematomal region (Yang et al., 2020). The concurrent effects of these events are issued in massive neuron apoptosis causing pejorative and sustained brain damage after ICH (Mohammed Thangameeran et al., 2020). However, unlike an ischemic stroke, there is no effective treatment for ICH. In this respect, an efficient and novel agent for preventing neuron death after ICH is necessary.
Neuron apoptosis occurs in ICH is the major reason for the poor prognosis following ICH injury (Zille et al., 2017). As a prominent cell type in the brain, astrocytes play an important role followed brain injury (Clarke and Barres, 2013). Several studies for protecting neurons by manipulating astrocytes have been revealed to be effective, and the role of astrocytes as both neuroprotective and exacerbating injury in ICH was reviewed recently (Barreto et al., 2011; Min et al., 2015). The reason astrocytes are used as therapeutic targets is that neurons cannot survive independently from astrocytes (Bylicky et al., 2018). Moreover, astrocyte viability is maintained stronger than neurons in the stroke. On this account, targeting surviving astrocytes offers an inestimable opportunity to recover the function of neurons and blood vessels in the process of ICH (Liu and Chopp, 2016). However, the explosive swelling of astrocyte contributes to its dysfunction and assistants with aquaporins (Thomas et al., 2004; Qiu et al., 2015). As a member of the aquaporins, aquaporin 4 (AQP4) is abundant in astrocytes processes and responsible for brain edema (Chu et al., 2016). Accumulating evidence has shown that inhibiting the expression of AQP4 could improve recovery of neurological function, against neuron apoptosis and inflammation response, while brain edema alleviated simultaneously (Tang and Yang, 2016). However, inhibitors for AQP4 have not been determined. Therefore, it is essential to explore an agent that could effectively regulate AQP4.
Glycyl-l-histidyl-l-lysine (GHK) is a kind of human tripeptide consist of GHK. It was first isolated by Pickart in 1973 that naturally occurring in plasma, saliva, and urine. Subsequent studies established that GHK and its copper (II)-chelated form (GHK-Cu) improved the process of regeneration, anti-inflammatory and anti-oxidant action (Pickart, 2008; Pickart et al., 2015). Furthermore, it has been convinced as a probable peptide for treating chronic obstructive pulmonary disease, attenuating skin inflammation, and inhibiting metastatic colon cancer (Gruchlik et al., 2012; Meiners and Eickelberg, 2012; Pickart et al., 2012). Also, studies have indicated that GHK enhances trophic factors secretion of mesenchymal stem cells, and exerts neuroprotective effects against neurodegenerative diseases by inhibiting inflammation, alleviating oxidative damage, even modulating the iron levels (Miller et al., 1990; Jose et al., 2014). Our previous study has found that GHK could alleviate neuron injury via VEGFA (Zhang H. et al., 2018). However, the effect and mechanism of GHK on ICH secondary injury which is related to glial cells is indecisive and should be further explored.
In this study, we studied the potential role of GHK in ICH rat models. We found that different concentrations of GHK could improve rats’ neurological function recovery, while alleviated brain edema, inflammation reaction, and down-regulated the level of AQP4 through miR-146a-3p after ICH. Furthermore, phosphatidylinositol 3-kinase (PI3K)/AKT pathway involves in the treatment of GHK in ICH. These results provide a novel therapeutic approach to ICH.
Materials and Methods
Adult male Wistar rats (between 250 and 280 g) were purchased from Changsheng Biotechnology Co., Ltd. Rats were carefully kept in an environmentally controlled room (22–25°C, 50% humidity) under a 12 h day-night rhythm. Food and water could be obtained ad libitum.
One hundred and twenty rats were randomly divided into five groups: (1) Sham group, 24 rats experienced the same surgical procedures as rats in the control group without collagenase VII injection. (2) Control group, 24 rats underwent the collagenase VII-induced ICH and injected vehicles intraperitoneally (i.p.) when the treatment groups were administered GHK. (3) GHK with 95% purity, was purchased from Chinapeptids Biotechnology Co., Ltd. (Zhou et al., 2017). GHK groups (GHK 1 μg/g, GHK 5 μg/g, and GHK 10 μg/g), rats in these groups were injected with GHK after the administration of collagenase VII. Twenty-four rats in each group were randomly divided into four groups by a researcher who was blind with the neurological deficits of these rats. Six rats were decapitated to obtain fresh brain tissue samples for water content. Six rats were perfused with fixative for histological preparation and analysis of the brains. Six rats were used for the neurological deficits scores until 7 days after ICH. Six rats were anesthetized by intraperitoneal injection of 2% pentobarbital intraperitoneally. Before they were decapitated, 300 mL physiological saline was administered to perform cardiac perfusion. A coronal cut was made with the pinhole as the center. The nasal side of perihematomal tissues in basal ganglia, which were prepared for biochemical analyses, were placed in liquid nitrogen for 30 min before storage at −80°C in enzyme-free tubes (Pei et al., 2016). All data were collected and analyzed by a researcher who was blind with the groups.
Collagenase VII-Induced Intracerebral Hemorrhage Model
Intracerebral hemorrhage was induced via stereotactic administration of 0.25 U/μl bacterial collagenase type VII (Sigma, United States), as Rosenberg described (Rosenberg et al., 1990). Rats in the GHK treatment group were intraperitoneally injected with GHK every 24 h dissolved in saline for 3 days after the administration of ICH, while those in the control group were treated with equal volumes of vehicle.
Brain Water Content
Rats were anesthetized, and the brains were collected 3 days following ICH. The wet weight of each sample was immediately measured by an electronic balance, following which the brains were dried at 100°C for 24 h to obtain the dry weight. Water content was calculated according to the following formula: [(wet weight – dry weight)/(wet weight) × 100 (%)] (Lee et al., 2008).
Evaluation of Neurological Deficits
Neurological deficits were evaluated using a five-point scale established by Rosenberg et al. (1990), The extent of circling of rats was graded from 0 (no circling) to 4 (always circled) at 3 and 7 days after ICH induction.
Immunohistochemistry and Nissl Staining
Immunohistochemistry experiments were performed using the UltraSensitiveTM SP (Mouse/Rabbit) immunohistochemistry Kit (Mixim, Fu Zhou, China), following the manufacturer’s instructions. The samples were fixed for 24 h by 4% paraformaldehyde and dehydrated using 70–100% alcohol. Paraffin-embedded brains were cut into 5 μm sections. The sections were suffered to antigen retrieval under high temperature and pressure conditions, following which they were treated with the endogenous peroxidase blockers and then blocked in serum for an hour. The sections were incubated in 1:200 AQP4 antibody overnight. Subsequently, the sections were incubated with secondary antibody (goat anti-rabbit), rinsed with phosphate buffer saline (PBS), stained with DAB, and hematoxylin and eosin (H&E) staining, dehydrated, transparentized, and mounted before microscopic analysis. We randomly selected 5 visual fields at the nasal side of perihematoma in basal ganglia, positive cells were discerned and analyzed by ImageJ software. For Nissl staining, the sections were subjected to cresyl violet acetate for 15 min at 23–25°C, rinsed in PBS, dehydrated, transparentized, and mounted before microscopic analysis.
The samples were fixed with 4% paraformaldehyde for 24 h and dehydrated using a 10–30% saccharose solution. OCT embedded brains were cut into 10 μm sections, the sections were rinsed three times with PBS, blocked for 1 h in 8% bovine serum albumin (BSA) dissolved in 0.01 M phosphate buffer (PB) containing 0.5% Triton-X100. Then incubated at 4°C overnight with anti-glial fibrillary acidic protein (GFAP) (cell signaling technology, 80788) diluted 1:100 in 0.01 M PB containing 1% BSA and 0.1% Triton-X100. After rinsing with PBS, all sections were incubated at room temperature for 2 h with IgG goat anti-rabbit (Thermo Fisher Scientific, Alexa Fluor 488) diluted 1:200 in 0.01 M PB containing 1% BSA and 0.3% Triton-X100. Nuclei were stained with 200 nM 4,6-diamidino-2-phenyl-indole (DAPI) for 5 min. Sections were viewed via a fluorescence microscope.
Cell Culture and in vitro Model of Hemorrhagic Toxicity
Astrocytes and 293T cells (Chinese Academy of Sciences, Shanghai) were cultured in dulbecco’s modified eagle medium (DMEM) (Gibco, UA) added 10% fetal bovine serum (Gibco, Australia), following which they were incubated at 100% humidity in an environment containing 5% CO2. To imitate hemorrhage toxicity in vitro, the model was constructed by applying 100 μM hemin to Astrocytes (Huang et al., 2002; Zille et al., 2017).
Synthetic RNA Oligonucleotides and Transfection
We purchased miRNA-146a-3p mimics, miRNA-146a-3p inhibitors, and a non-sense sequence for use as a miRNA negative control (NC) from Genepharma (Shanghai, China) (Supplementary Table 2). Astrocytes were transfected with Lipofectamine 2000 (Invitrogen, United States), according to the manufacturer’s instructions.
RNA Extraction and Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted from cellular and cerebral samples using TRIzol reagent (Invitrogen, Carlsbad, CA, United States). Reverse transcription of miRNA was performed using the Hairpin-it miRNAs qPCR Quantitation Kit (GenePharma, Shanghai, China), following the manufacturer’s protocol. T ABI 7500 system (Thermo Fisher Scientific, Carlsbad, CA, United States) was using for real-time PCR. The relative miRNA expression of each gene was normalized to the expression of U6 RNA. The primers were synthesized by Sangon Biotech (Shanghai). All primer sequences are listed in Supplementary Table 1.
Western Blot Analysis
Cellular and cerebral samples were lysed in RIPA (Beyotime, China) containing inhibitor cocktail (Roche, Germany) for 20 min, following which the lysates were centrifuged (12,000 × g) at 4°C for 15 min. Protein concentration was determined using a BCA Protein Kit (Thermo, Rockford, IL, United States). Sample lysates were separated via 4–14% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), following which the proteins were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were then incubated in 1% bovine serum albumin (BSA) solution with the following primary antibodies overnight at 4°C: anti-AQP4 (Abcam, ab18956), anti-MMP2 (Abcam, ab37150), anti-MMP9 (Abcam, ab38898), anti-TIMP1 (Abcam, ab109125) and anti-beta actin (Abcam, ab8227). The membranes were then incubated with secondary antibody for 1 h at room temperature. We analyzed the relative signal densities by ImageJ software, and β-actin density was used as the internal control.
Cell Counting Kit-8 (CCK-8) Assay for Cell Viability and Cell Proliferation
Cell viability was determined using the CCK-8 solution (Beyotime, China), under the manufacturer’s instructions. Approximately 5 × 103 cells were seeded in each well of a 96-well plate overnight, following which the cells were applied hemin for 18 h, GHK was added to the culture medium simultaneously. Cell proliferation detection was according to the manufacturer’s instruction. In brief, 2 × 103 cells were seeded in each well of a 96-well plate for 4 h, then GHK was added and this time is known as 0 h, and the cells were cultured for 24 and 48 h for detecting cell proliferation. A total of 10 μl of CCK-8 solution was added to each well, and the cells were incubated for 1 h in the incubator. The absorbance of each well was quantified at 450 nm using an automated ELISA reader (SpectraMax® M5, Molecular Devices, United States). Cell viability was calculated as follows: (A450 of experimental wells/A450 of normal wells) × 100%.
Lactate Dehydrogenase (LDH) Assay for Cytotoxicity
Cytotoxicity was determined using an LDH Assay Kit (Beyotime, China), in accordance with the manufacturer’s instructions. Following applying hemin for 18 h, the supernatant was collected and transferred to 96-well plates to detect LDH release. The absorbance of each sample was measured at 490 nm using an automated ELISA reader (SpectraMax® M5, Molecular Devices, United States). Percent cytotoxicity was calculated as follows: (A490 of experimental culture medium-A490 of normal medium)/(A490 of maximum LDH release-A490 of normal wells) × 100%.
Measurement of the TNF-α and IL-1β by ELISA
The TNF-α and IL-1β levels in the perihematomal were determined using ELISA kits (USCN Life Science Inc., Wuhan, China) according to the manufacturer’s instructions. The absorbance of each sample was quantified at 450 nm using an automated ELISA reader (SpectraMax® M5, Molecular Devices, United States).
The pmirGLO Dual-Luciferase miRNA target expression vectors were constructed by Genepharma (Shanghai, China). 1 × 104 293T cells were seeded in each well of a 24-well plate overnight, following which they were co-transfected with wild-type and mutant AQP4 promoter-luciferase plasmids (0.1 μg per well) and 0.4μg miR-146a-3p mimics or NCs using Lipofectamine 2000 (Invitrogen, United States). The pmirGLO-wt-AQP4 and pmirGLO-mt-AQP4 were purchased from Genepharma Transfection efficiency was standardized based on that of TK activity. Luciferase activity was quantified using a dual-luciferase assay system (Promega, E1910).
Each assay was repeated independently at least three times to ensure the reliability of this study and the measurement data are expressed as the mean ± SD. We also performed a prior power analysis using the G∗Power 18.104.22.168 software with a significance level of 5% and a power level > 0.9 to ensure we used an adequate number of animals per group. All data were analyzed using SPSS 23.0 software (IBM Corp., Armonk) and Graphpad Prism 5.0 (GraphPad Software). Comparisons among multiple groups were performed using one-way ANOVA followed by a Bonferroni or SNK test. Behavioral data were analyzed using the Kruskal–Wallis test with Dunn’s test for multiple comparisons. Coefficients of correlation were analyzed by the Pearson correlation method. The demarcation of statistical significance was set at p < 0.05.
GHK Alleviated Neurological Deficits and Promoted Neuron Survival Following ICH
To investigate the potential role of GHK post-ICH, we isolated the rat brain 3 days after ICH. We measured brain water content and intact neuron number in different groups of rats. Neurological deficits were detected 3 and 7 days after ICH. Our results indicated that GHK significantly decreased brain water content (Figure 1A) and relieved the neurological deficits in ICH rats (Figure 1B). Numbers of survival neurons were significantly increased in rats treated with GHK, relative to findings observed in control rats (Figures 1C,D).
Figure 1. GHK alleviated neurological injury and neuronal apoptosis following intracerebral hemorrhage (ICH). (A) GHK delivery via the intraperitoneal route decreased water content in the brain 72 h after ICH. (B) Analyses of neurological deficits revealed that treatment with both 5 μg/g and 10 μg/g GHK facilitated neurological recovery at 3 and 7 days after ICH. (C) Nissl staining in vivo. Paraffin slices (5 μm) from the control and GHK groups were stained with cresyl violet acetate. (arrows are pointing the intact neurons). (D) The number of the intact neuron. (n = 6. #p < 0.05 vs. sham; *p < 0.05 vs. control; **p < 0.01 vs. control, ##p < 0.01 vs. sham).
GHK Ameliorated Inflammation and Metalloprotease/Anti-metalloprotease Imbalance in ICH
To investigate the effect of GHK following ICH, we estimated the level of TNF-α and IL-1β in ICH brain samples using ELISA (Figures 2A,B). The expression of MMP2, MMP2, TIMP1 was detected using western blot (Figures 2C–E). The result has shown that GHK significantly decreased the expression of TNF-α and IL-1β, which are the crucial factors in the inflammation of ICH. Furthermore, the expression of MMP2 and MMP9 downregulated while the expression of TIMP1 increased. These findings demonstrated that GHK could alleviate the inflammation and reverse the imbalance of metalloprotease/anti-metalloprotease following ICH. However, the activation of metalloproteases is related to astrocytes injury in ICH.
Figure 2. GHK alleviated inflammation and metalloprotease/anti-metalloprotease imbalance in ICH. (A) The expression level of TNF-α. (B) The expression level of IL-1β. (C–E) The expression of MMP2, MMP9, and TIMP1 in different groups of rats. (n = 6. #p < 0.05 vs. sham; *p < 0.05 vs. control; **p < 0.01 vs. control).
GHK Alleviated Astrocytes Injury Following ICH and Down-Regulated AQP4 Expression
Astrocytes are the major glial cells in the brain and charge of brain homeostasis maintenance. To investigate the effect of GHK on astrocytes following ICH, we evaluated the cell viability and cell proliferation in vitro by CCK-8 (Figures 3A,C), cytotoxicity by Lactate dehydrogenase (LDH) (Figure 3B). We also detected the activation of astrocytes around the hematoma, the expression of GFAP without significant difference (Supplementary Figure 2). The results showed that GHK significantly increased the survival of astrocytes, but no significant difference in proliferation. Moreover, we detected the expression of AQP4 using immunohistochemistry (Figures 4A,B) and western blot (Figures 4C,D). The results determined the level of AQP4 decreased following the increase of GHK concentration.
Figure 3. GHK improved the survival of astrocytes. (A) GHK increased the viability of astrocytes in dose-dependent manner. (B) GHK decreased the cytotoxicity of astrocyte. (C) GHK not significantly promoted astrocytes proliferation. (One-way ANOVA was used for repeated measurements. Data are shown as the mean ± SD, n = 3. *p < 0.05 vs. control; **p < 0.01 vs. control; ▲▲p < 0.05 vs. GHK 5 μM).
Figure 4. GHK down-regulated the expression of AQP4. (A) Immunohistochemistry analysis of AQP4 expression. Paraffin slices (5 μm) from the control and GHK groups were stained with AQP4. (B) Quantification of AQP4 positive cells (n = 6. #p < 0.05 vs. sham; *p < 0.05 vs. control; **p < 0.01 vs. control). (C) Western blot analysis illustrating the effect of GHK on AQP4 expression in rats perihematomal tissue (n = 6. #p < 0.05 vs. sham; *p < 0.05 vs. control; **p < 0.01 vs. control). (D) The expression of AQP4 in astrocytes under hemin-stimulation. (One-way ANOVA was used for repeated measurements. Data are shown as the mean ± SD, n = 3. *p < 0.05 vs. control; **p < 0.01 vs. control; ▲p < 0.05 vs. GHK 5μM).
AQP4 Was a Functional Target Gene of miRNA-146a-3p
To investigate the reason of AQP4 decreased after GHK treatment, we predict the potential target microRNAs (miRNAs) of AQP4 (Supplementary Table 1) and got intersection elements with miRNAs down-regulated in ICH patients (Zhu et al., 2015; Figure 5A). Then we detected the expression of miR-146-3p in astrocytes using real-time PCR. The result demonstrated that miR-146a-3p significantly upregulated followed GHK treatment (Figures 5B,C). To confirm the prediction, we found the binding region of miR-146a-3p and AQP4 (Figure 5F) and transfected miR-146a-3p into astrocytes. Before the experiment, the expression level was detected by real-time PCR (Figure 5D). We found that miR-146a-3p expression revealed a completely contrary trend to AQP4 expression (Figure 5E). Furthermore, dual-luciferase assays were employed to illuminate the interaction of miR-146a-3p with AQP4 mRNA. A significant decrease in the relative luciferase activity was observed in the miR-146a-3p + pGLO-wt-AQP4 group compared to the other groups (p < 0.05) (Figure 5G).
Figure 5. AQP4 was a functional target gene of miR-146a-3p. (A) The intersection of potential miRNAs of AQP4 and miRNAs downregulated in ICH patients. (B) Level of miR-146a-3p in different concentration GHK-treated ICH rat perihematomal tissue, relative to control values (n = 6, **p < 0.01 vs. control). (C) RT-qPCR assays for miR-146a-3p in astrocytes under hemin-stimulation as compared with control group (n = 3, **p < 0.01). (D) The expression level of miR-146a-3p after mimics, inhibitor and NCs transfected (n = 3, *p < 0.05, **p < 0.01). (E) The expression of AQP4 following miR-146a-3p mimics, inhibitor and NCs transfected. (F) Binding sites for miR-146a-3p and AQP4. (G) Levels of luciferase activity in 293T cells co-transfected with miR-146a-3p and AQP4. *p < 0.05.
GHK Increases miR-146a-3p Levels Through the PI3K/Akt Pathway and miR-146a-3p Inhibitor Reverses GHK-induced AQP4 Reduction in Astrocytes.
Previous studies have demonstrated that the PI3K/Akt pathway plays important role in ICH injury. Cui et al. (2015, 2017) has revealed that the activation of the PI3K/Akt pathway acted a protective effect in ICH. Also, some researchers have demonstrated inhibiting PI3Kleaded to a dose-dependent downregulation in the expression of mature miR-146a (Jiang, 2014). To study the pathway of which GHK increase miR-146a-3p levels, different concentrations of PI3K/Akt inhibitor added to the medium of astrocytes, which were stimulated with 10 μM GHK simultaneously (Figure 6A). We revealed that various concentrations of the PI3K inhibitor LY294002 and Akt inhibitor MK-2206 2HCL suppressed the up-regulation of miR-146a-3p levels induced by GHK in a concentration-dependent manner (Figures 6B,C). Furthermore, miR-146a-3p inhibitors can reverse the downregulation of AQP4 (Figure 6D). These results indicated that GHK decreases AQP4 expression via miR-146a-3p through the PI3K/Akt pathway (Figure 7).
Figure 6. GHK increases miR-146a-3p levels through the PI3K/Akt pathway and miR-146a-3p inhibition reverses AQP4 decrease induced by GHK in astrocytes. (A) Expression of p-Akt and Akt in astrocytes with different concentration of GHK (n = 3, *p < 0.05, **p < 0.01). (B,C) Expression of miR-146a-3p in astrocytes with PI3K inhibitor LY294002 and Akt inhibitor MK-2206 2HCL were incubated with 10 μM GHK simultaneously (n = 3. *p < 0.05 vs. GHK. Analysis using ImageJ). (D) Protein levels of AQP4 in astrocytes treated with GHK and the miR-146a-3p NC or inhibitor. (n = 3, *p < 0.05. Analysis using ImageJ).
Figure 7. Summary model of the down-regulation of AQP4 induced by GHK via Akt/miR-146a-3p/AQP4 in astrocytes.
GHK, a human tripeptide, natural and non-toxic, exhibits extraordinary effects with anti-inflammatory and neuroprotective effects in acute lung injury (Pickart et al., 2014; Park et al., 2016) and neurodegenerative diseases (Pickart et al., 2012, 2017). In our study, GHK protected against ICH injury by reducing brain edema and promoting neuron survival. Surprisingly, we found that intraperitoneally injected at a dose of 10 μg/g significantly alleviated neurological deficits until 7 days post-ICH, which suggested a dose of 10 μg/g can also play a role in the subacute phase post-ICH. These findings provided further evidence that GHK could be an available agent for ICH therapy. Our results demonstrated novel insights into the potential roles of GHK ICH secondary injury.
Intracerebral hemorrhage injury mainly results from the secondary injury followed hematoma formation including hemoglobin cytotoxicity and explosive inflammatory response, which mainly resulted in brain edema and neuron death (Shi et al., 2012; Zhang et al., 2014). Therefore, timely control of cytotoxic edema may contribute to alleviating hemorrhagic injury. We observed that GHK significantly alleviated the brain edema, neurological deficits, improved neuron survival and reduced the level of TNF-α and IL-1β. Furthermore, we found that GHK relieved the imbalance of matrix metalloproteinase (MMPs) synchronously. According to the previous study, the matrix metalloproteinase level was clearly evaluated after ICH, and the activation of MMP9 in astrocytes exacerbated ICH (Lattanzi et al., 2020). We believe that GHK may be an efficient agent for ICH by targeting astrocytes.
Accumulating evidence revealed that astrocytes play various homeostatic roles in the brain (Sofroniew and Vinters, 2010; Sticozzi et al., 2013). The activity of astrocytes is an important basis for the survival of neurons in brain injury (Ouyang et al., 2014). In addition to building the blood-brain barrier and secreting neural trophic factors, astrocytes are also responsible for brain edema and inflammation response after ICH (Chen-Roetling et al., 2015). In this study, we found that GHK could increase the viability and alleviate the cytotoxicity of astrocytes. These results showed that GHK significantly alleviated the injury of astrocytes due to ICH. Aquaporins play important roles in neural injury (Wang et al., 2015). AQP4 is one of the key transport channels in the central nervous system, which is abundant in astrocytes foot processes (Bonomini and Rezzani, 2010). Previous studies have demonstrated that AQP4 reactivity increase following ICH which plays a critical role in brain edema and cytotoxic cell death (Appelboom et al., 2015; Qiu et al., 2015). Increasing evidence indicates that inhibition of AQP4 alleviates the brain injury and improves the recovery of ICH (Zhong et al., 2013; Wang et al., 2015). We observed that the expression of AQP4 significantly declined with GHK treatment. However, the mechanism of GHK regulating AQP4 should be further explored.
Several studies of stroke have demonstrated that signal pathways regulate AQP4 expressions, such as the MAPK pathway and NF-κB pathway (Qi et al., 2011; Boldin and Baltimore, 2012). Moreover, several miRNAs, such as miR-29b and miR-130a, regulate AQP4 expression in ischemic stroke. miRNAs serve as an important constituent of non-coding RNAs. They bind to complementary sequences on target mRNAs, resulting in negative regulation through degraded target mRNA or repressed the translation (Beermann et al., 2016). Our previous results demonstrated that miR-146a-3p significantly decreased in ICH patients’ peripheral blood (Zhu et al., 2015). Ji et al. (2013) demonstrated that miR-146a regulated the level of SOD in H2O2 stimulated PC 12 cells. In addition, miR-146a-3p has been widely known as an important molecule in the astrocyte-related inflammatory response in neuropathy such as Alzheimer’s Disease and epilepsy (Aronica et al., 2010; Cui et al., 2010; Iyer et al., 2012). We found that AQP4 was a functional target gene of miR-146a-3p, and the level of miR-146a-3p was completely contrary to AQP4 following GHK treatment. Furthermore, the dual-luciferase activity of miR-146a-3p and AQP4 was significantly down-regulated, which indicated the direct effect of miR-146a-3p with AQP4. These results suggested that miR-146a-3p plays a role in GHK regulation of AQP4.
Accumulating evidence has indicated that the activation of the PI3K/Akt pathway involves the improvement of neurological functional recovery on ICH (Niu and Hu, 2017; Zhang W. et al., 2018). Jiang (2014) showed that PI3K/Akt pathway activation promoted the expression of miR-146a-3p in human alveolar epithelial cells, Walsh et al. (2015) reported that PI3K/Akt pathway involves in the regulation of miR-146a in T-cells. Moreover, existing evidence demonstrated that inhibiting the phosphorylation of PI3K could increase AQP4 expression, which suggested that PI3K/Akt involves in the regulation of AQP4 (Song et al., 2018). In this study, we observed that GHK increased the phosphorylation of Akt in astrocytes. Moreover, GHK upregulated the level of miR-146a-3p and decreased the expression of AQP4, while the LY294002 and MK-2206 2HCL could reverse these effects. Therefore, we assumed that the PI3K/Akt pathway was involved in the GHK-induced upregulation of miR-146a-3p and that the miR-146a-3p/AQP4 axis played a role in manipulating astrocytes following ICH injury.
Some limitations in our study must be documented. We used collagenase VII induced ICH model in this study which cannot completely simulate the pathophysiological process of ICH. We found that GHK could increase the phosphorylation of PI3K/Akt pathway, but we are not focus on the directly binding component. However, we focus on the effect of GHK on astrocytes only, rather than the interaction with neurons. Therefore, the specific role of GHK on ICH should be further explored.
In conclusion, our findings demonstrated that GHK reduced brain edema, improved neurological recovery, and alleviated the inflammation in a rat model of ICH. In addition, we observed that GHK treatment downregulated AQP4 expression and decreased injury of astrocytes, likely via the upregulation of miR-146a-3p. Moreover, inhibition of the PI3K/Akt activation restrained the GHK-induced upregulation of miR-146a-3p and the downregulation of AQP4. Our findings suggested that GHK may become a novel therapeutic for ICH.
Data Availability Statement
All datasets generated for this study are included in the article/Supplementary Material.
All procedures were conducted in accordance with the regulations of the animal protection laws of China and approved by the Animal Ethics Committee of China Medical University (2012-38-1 and 2017008).
HZ: data curation, investigation, methodology, writing original draft, investigation. YW: data curation formal analysis, methodology, conceptualization. LL: visualization. CZ: writing—review and editing. ZH: funding acquisition, conceptualization, supervision, writing—review and editing. All authors contributed to the article and approved the submitted version.
This research was funded by the National Natural Science Foundation of China (81571120). This study was supported by grants from the Southern China International Cooperation Base for Early Intervention and Functional Rehabilitation of Neurological Diseases (2015B050501003), Guangdong Provincial Engineering Center For Major Neurological Disease Treatment, Guangdong Provincial Translational Medicine Innovation Platform for Diagnosis and Treatment of Major Neurological Disease, Guangdong Provincial Clinical Research Center for Neurological Diseases.
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We acknowledge the support from the Department of Developmental Cell Biology, Cell Biology Division, Key Laboratory of Cell Biology, Ministry of Public Health, Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University.
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnins.2020.576389/full#supplementary-material
Supplementary Figure 1 | The level of phosphorylate-Akt in brain tissue detected by western blotting.
Supplementary Figure 2 | The expression of GFAP around the hematoma.
Supplementary Figure 3 | Inhibition of AQP4 in astrocytes improve the survival of neurons.
Supplementary Figure 4 | The expression of AQP4 in astrocytes treated with GHK and miR-146a-3p NC or mimics.
Supplementary Table 1 | The target miRNAs of AQP4.
Supplementary Table 2 | The Sequences of RNA oligos.
Supplementary Table 3 | The sequences of primers.
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Aronica, E., Fluiter, K., Iyer, A., Zurolo, E., Vreijling, J., van Vliet, E. A., et al. (2010). Expression pattern of mir-146a, an inflammation-associated microrna, in experimental and human temporal lobe epilepsy. Eur. J. Neurosci. 31, 1100–1107. doi: 10.1111/j.1460-9568.2010.07122.x
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Cui, J., Cui, C., Cui, Y., Li, R., Sheng, H., Jiang, X., et al. (2017). Bone marrow mesenchymal stem cell transplantation increases gap-43 expression via erk1/2 and pi3k/akt pathways in intracerebral hemorrhage. Cell. Physiol. Biochem. 42, 137–144. doi: 10.1159/000477122
Cui, J. G., Li, Y. Y., Zhao, Y., Bhattacharjee, S., and Lukiw, W. J. (2010). Differential regulation of interleukin-1 receptor-associated kinase-1 (irak-1) and irak-2 by microrna-146a and nf-kappab in stressed human astroglial cells and in alzheimer disease. J. Biol. Chem. 285, 38951–38960. doi: 10.1074/jbc.M110.178848
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Huang, P., Freeman, W. D., Edenfield, B. H., Brott, T. G., Meschia, J. F., and Zubair, A. C. (2019). Safety and efficacy of intraventricular delivery of bone marrow-derived mesenchymal stem cells in hemorrhagic stroke model. Sci. Rep. 9:5674. doi: 10.1038/s41598-019-42182-1
Iyer, A., Zurolo, E., Prabowo, A., Fluiter, K., Spliet, W. G., van Rijen, P. C., et al. (2012). Microrna-146a: a key regulator of astrocyte-mediated inflammatory response. PLoS One 7:e44789. doi: 10.1371/journal.pone.0044789
Jiang, X. (2014). Different signal pathways regulate il-1beta-induced mature and primary mirna-146a expression in human alveolar epithelial cells. Acta Physiol. Hung. 101, 282–290. doi: 10.1556/APhysiol.101.2014.3.3
Jose, S., Hughbanks, M. L., Binder, B. Y., Ingavle, G. C., and Leach, J. K. (2014). Enhanced trophic factor secretion by mesenchymal stem/stromal cells with glycine-histidine-lysine (ghk)-modified alginate hydrogels. Acta Biomater. 10, 1955–1964. doi: 10.1016/j.actbio.2014.01.020
Lee, S. T., Chu, K., Jung, K. H., Kim, S. J., Kim, D. H., Kang, K. M., et al. (2008). Anti-inflammatory mechanism of intravascular neural stem cell transplantation in haemorrhagic stroke. Brain 131, 616–629. doi: 10.1093/brain/awm306
Meiners, S., and Eickelberg, O. (2012). Next-generation personalized drug discovery: the tripeptide ghk hits center stage in chronic obstructive pulmonary disease. Genome Med. 4:70. doi: 10.1186/gm371
Miller, D. M., DeSilva, D., Pickart, L., and Aust, S. D. (1990). Effects of glycyl-histidyl-lysyl chelated cu(ii) on ferritin dependent lipid peroxidation. Adv. Exp. Med. Biol. 264, 79–84. doi: 10.1007/978-1-4684-5730-8_11
Min, H., Hong, J., Cho, I. H., Jang, Y. H., Lee, H., Kim, D., et al. (2015). Tlr2-induced astrocyte mmp9 activation compromises the blood brain barrier and exacerbates intracerebral hemorrhage in animal models. Mol. Brain 8:23. doi: 10.1186/s13041-015-0116-z
Mohammed Thangameeran, S. I., Tsai, S.-T., Hung, H.-Y., Hu, W.-F., Pang, C.-Y., Chen, S.-Y., et al. (2020). A role for endoplasmic reticulum stress in intracerebral hemorrhage. Cells 9:750. doi: 10.3390/cells9030750
Niu, J., and Hu, R. (2017). Role of flunarizine hydrochloride in secondary brain injury following intracerebral hemorrhage in rats. Int. J. Immunopathol. Pharmacol. 30, 413–419. doi: 10.1177/0394632017742224
Ouyang, Y. B., Xu, L., Yue, S., Liu, S., and Giffard, R. G. (2014). Neuroprotection by astrocytes in brain ischemia: importance of micrornas. Neurosci. Lett. 565, 53–58. doi: 10.1016/j.neulet.2013.11.015
Park, J. R., Lee, H., Kim, S. I., and Yang, S. R. (2016). The tri-peptide ghk-cu complex ameliorates lipopolysaccharide-induced acute lung injury in mice. Oncotarget 7, 58405–58417. doi: 10.18632/oncotarget.11168
Pei, H., Jiang, T., Liu, G., Li, Z., Luo, K., An, J., et al. (2016). The effect of minimally invasive hematoma aspiration on the jnk signal transduction pathway after experimental intracerebral hemorrhage in rats. Int. J. Mol. Sci. 17:710. doi: 10.3390/ijms17050710
Pickart, L., Vasquez-Soltero, J. M., and Margolina, A. (2012). The human tripeptide ghk-cu in prevention of oxidative stress and degenerative conditions of aging: implications for cognitive health. Oxid. Med. Cell. Longev. 2012:324832. doi: 10.1155/2012/324832
Pickart, L., Vasquez-Soltero, J. M., and Margolina, A. (2015). Ghk peptide as a natural modulator of multiple cellular pathways in skin regeneration. BioMed Res. Int. 2015:648108. doi: 10.1155/2015/648108
Pickart, L., Vasquez-Soltero, J. M., and Margolina, A. (2017). The effect of the human peptide ghk on gene expression relevant to nervous system function and cognitive decline. Brain Sci. 7:20. doi: 10.3390/brainsci7020020
Qi, L. L., Fang, S. H., Shi, W. Z., Huang, X. Q., Zhang, X. Y., Lu, Y. B., et al. (2011). Cyslt2 receptor-mediated aqp4 up-regulation is involved in ischemic-like injury through activation of erk and p38 mapk in rat astrocytes. Life Sci. 88, 50–56. doi: 10.1016/j.lfs.2010.10.025
Qiu, G. P., Xu, J., Zhuo, F., Sun, S. Q., Liu, H., Yang, M., et al. (2015). Loss of aqp4 polarized localization with loss of beta-dystroglycan immunoreactivity may induce brain edema following intracerebral hemorrhage. Neurosci. Lett. 588, 42–48. doi: 10.1016/j.neulet.2014.12.053
Shi, B. Z., Meng, X. F., Yang, J. X., Hao, X. W., Cui, T., Lu, Z. F., et al. (2012). Effect of erythrocytes on brain water content and haem oxygenase-1 expression in rats with traumatic intracerebral haemorrhage. Acta Neurochir. 154, 1081–1086. doi: 10.1007/s00701-012-1335-z
Song, D., Liu, X., Diao, Y., Sun, Y., Gao, G., Zhang, T., et al. (2018). Hydrogenrich solution against myocardial injury and aquaporin expression via the pi3k/akt signaling pathway during cardiopulmonary bypass in rats. Mol. Med. Rep. 18, 1925–1938. doi: 10.3892/mmr.2018.9198
Sticozzi, C., Belmonte, G., Meini, A., Carbotti, P., Grasso, G., and Palmi, M. (2013). Il-1beta induces gfap expression in vitro and in vivo and protects neurons from traumatic injury-associated apoptosis in rat brain striatum via nfkappab/ca(2)(+)-calmodulin/erk mitogen-activated protein kinase signaling pathway. Neuroscience 252, 367–383. doi: 10.1016/j.neuroscience.2013.07.061
Thomas, R., Salter, M. G., Wilke, S., Husen, A., Allcock, N., Nivison, M., et al. (2004). Acute ischemic injury of astrocytes is mediated by na-k-cl cotransport and not ca2+ influx at a key point in white matter development. J. Neuropathol. Exp. Neurol. 63, 856–871. doi: 10.1093/jnen/63.8.856
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Wang, B. F., Cui, Z. W., Zhong, Z. H., Sun, Y. H., Sun, Q. F., Yang, G. Y., et al. (2015). Curcumin attenuates brain edema in mice with intracerebral hemorrhage through inhibition of aqp4 and aqp9 expression. Acta Pharmacol. Sin. 36, 939–948. doi: 10.1038/aps.2015.47
Yang, G.-Q., Huang, J.-C., Yuan, J.-J., Zhang, Q., Gong, C.-X., Chen, Q., et al. (2020). Prdx1 reduces intracerebral hemorrhage-induced brain injury via targeting inflammation- and apoptosis-related mrna stability. Front. Neurosci. 14:181. doi: 10.3389/fnins.2020.00181
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Zhang, W., Cui, Y., Gao, J., Li, R., Jiang, X., Tian, Y., et al. (2018). Recombinant osteopontin improves neurological functional recovery and protects against apoptosis via pi3k/akt/gsk-3beta pathway following intracerebral hemorrhage. Med. Sci. Monit. 24, 1588–1596. doi: 10.12659/MSM.905700
Zhang, Z., Liu, Y., Huang, Q., Su, Y., Zhang, Y., Wang, G., et al. (2014). Nf-kappab activation and cell death after intracerebral hemorrhage in patients. Neurol. Sci. 35, 1097–1102. doi: 10.1007/s10072-014-1657-0
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Zhou, X. M., Wang, G. L., Wang, X. B., Liu, L., Zhang, Q., Yin, Y., et al. (2017). Ghk peptide inhibits bleomycin-induced pulmonary fibrosis in mice by suppressing tgfβ1/smad-mediated epithelial-to-mesenchymal transition. Front. Pharmacol. 8:904. doi: 10.3389/fphar.2017.00904
Zhu, Y., Wang, J. L., He, Z. Y., Jin, F., and Tang, L. (2015). Association of altered serum micrornas with perihematomal edema after acute intracerebral hemorrhage. PLoS One 10:e0133783. doi: 10.1371/journal.pone.0133783
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Keywords: intracerebral hemorrhage, glycine-histidine-lysine, astrocytes, miR-146a-3p, AQP4
Citation: Zhang H, Wang Y, Lian L, Zhang C and He Z (2020) Glycine-Histidine-Lysine (GHK) Alleviates Astrocytes Injury of Intracerebral Hemorrhage via the Akt/miR-146a-3p/AQP4 Pathway. Front. Neurosci. 14:576389. doi: 10.3389/fnins.2020.576389
Received: 26 June 2020; Accepted: 03 September 2020;
Published: 28 October 2020.
Edited by:Arumugam R. Jayakumar, Miami VA Healthcare System, United States
Reviewed by:Abdeljalil Elgot, Hassan Premier University, Morocco
Kodisundaram Paulrasu, University of Miami, United States
Usha Nagarajan, Central University of Haryana (CUH), India
Copyright © 2020 Zhang, Wang, Lian, Zhang and He. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.


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Viral News Boy :- Maintaining a healthy relationship is a skill that takes time to master. We understand how to begin connecting and conversation. However, not everyone understands how to save a relationship, make it grow, and move forward with your spouse. Above all, not everyone recognizes when it’s time to end a poor relationship. When it comes to dating a man, why do women overlook critical red flags? After years of constant contact, how could couples end up unhappy? Why do women leave men they love, and vice versa? Because it’s difficult to pinpoint the source of the problem, we’ve decided to take a different approach.
Every woman needs to know that her man will be there for her. Males and females both have a dread of the unknown. It hurts to not know what’s going on behind closed doors. Anticipation causes anxiety, and a situational relationship causes a lot of stress. Nobody wants to be a backup plan for someone. Your partner wants you to provide her stability and if that can’t be provided no matter how much she loves you. She will leave .
2. They feel underappreciated and ignored.
Women leave males for a variety of reasons, the most common of which is a sense of neglect. Relationships provide three basic requirements for people: love, support, and attention. When a spouse believes they aren’t getting these things, they become irritated and frequently leave. Because they are unhappy in their relationships, women are more inclined to cheat. When you are dating someone cultivate an habit of appreciating your partner. Believe me it really goes a long way. It is not easy for someone to love you with your flaws and vice versa, so always appreciate your partner for loving you.
3. Selflessness has taken control
You put more time into activities that you enjoy, and the less time you offer her, the more warped her perception of the relationship becomes. ladies don’t want to be regarded as an afterthought; they want to be treated as a priority. If you ask to be left alone, she will first feel reluctant, but over a period of time, she will genuinely contemplate leaving you alone permanently. In a relationship, there is no “I.” the word “I” is the most selfish word in the world so you should avoid it.
4. You are immature.
A woman wants a man she can respect, not one who hasn’t grown out of his immature behaviors. A lady seeks a man who is capable of carrying out his commitments. There’s nothing great about having a man who still makes bad mistakes with his pals or seeks his mother’s advice on everything he does. Long-term relationships necessitate maturity, which a woman seeks in her spouse. You are not children whereby you argue or fight everyday. Couple should be able to understand themself and if there is lack of understanding due to your immature behavior she is likely to leave you for someone else.
5. There are just too many lies in your relationship.
Every now and then, everyone lies. Is it OK to lie? No, It is not appropriate to lie to the person you love; your relationship is built on the trust you have, and lying to your partner means throwing that trust out the window. They will soon discover that you lied in some way, and your relationship will come to an end as a result (or at least, the trust they had for you will never be the same). You’ll lie to someone again if you’ve once lied to them. Relationship built on lies doesn’t survive.
6. The love may still be there,but the attraction has gone.
This is a difficult one to convey to someone who has never gone through it, but it can be one of the main reasons why a woman leaves a man she claims to love. It’s entirely possible to love someone while not being attracted to them. This can take a long time and is typically a gradual procedure. It’s possible they’re not the person you fell in love with at first. We all change as we go through life, but some of us evolve at different rates or in altogether different ways than our partners. You should even be grateful she left, when the love is not there anymore the best thing one could do is leave the relationship.
7. Trust issues
This is a difficult one, but it’s also a very common one. One of the most fundamental parts of a relationship, and possibly the most important for it to work, is trust. We all want in trust in our relationships, but what happens when we are unable or unwilling to do so? Lack of trust can stifle a lot of the other wonderful aspects of being in a relationship,compatibility, attractiveness, understanding, and fun. You may be in the ideal relationship if you can cross all of these things off a list, but there is no actual foundation for your relationship if you don’t trust each other. Women leave the person they love when they discovered that he can’t be trusted. What is the essence of being in a relationship with someone you can’t trust. Partners should be able to trust themselves. when there is trust issues the best is to leave the relationship. Laise can’t marry who they cannot trust.

domain: sociology


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Comparing the 1965 Ford Mustang Shelby GT350 and the 2020 Ford Mustang Shelby GT350R involves contrasting two iconic models from different eras. While both are part of the Mustang Shelby lineage, they differ significantly in terms of design, technology, performance, and the automotive landscape of their respective times. Here’s a comparison between the two:
1. Era and Design:
- 1965 Shelby GT350: The 1965 GT350 was based on the first-generation Mustang platform. It had a classic, retro-inspired design with a distinctive fastback body style. It featured subtle modifications like a unique front grille, side scoops, and racing stripes, showcasing its performance-oriented nature for the time.
- 2020 Shelby GT350R: The 2020 GT350R is based on the sixth-generation Mustang platform. It boasts a modern and aggressive design with aerodynamic enhancements, a large front grille, functional hood vents, and a rear spoiler. The overall look is more aggressive and track-focused, reflecting contemporary design trends.
- 1965 Shelby GT350: The original GT350 was powered by a naturally aspirated 4.7-liter V8 engine that produced around 306 horsepower. It was designed to be a high-performance road and track car, with suspension and handling upgrades.
- 2020 Shelby GT350R: The 2020 GT350R is powered by a naturally aspirated 5.2-liter V8 engine known as the “Voodoo” engine. It delivers 526 horsepower and is mated to a six-speed manual transmission. The GT350R features advanced aerodynamics, magnetic ride suspension, and other performance-focused components that make it a potent track machine.
- 1965 Shelby GT350: The technology available in the 1965 GT350 was representative of its time, with basic mechanical components and limited electronic features.
- 2020 Shelby GT350R: The 2020 GT350R benefits from advanced technology, including electronic aids like traction control and various driving modes that allow the driver to tailor the car’s performance characteristics to different conditions.
4. Handling and Dynamics:
- 1965 Shelby GT350: The handling of the 1965 GT350 was enhanced for its time, but it lacked the advanced suspension and chassis technologies found in modern sports cars.
- 2020 Shelby GT350R: The 2020 GT350R’s handling benefits from modern engineering advancements, including a specially tuned suspension, lightweight components, and advanced stability control systems.
5. Rarity and Collectibility:
- 1965 Shelby GT350: The 1965 GT350 is a highly sought-after classic car with historical significance, making it valuable and collectible among enthusiasts and collectors.
- 2020 Shelby GT350R: The 2020 GT350R is a limited-production model, which also contributes to its desirability among modern muscle car enthusiasts.
In summary, the 1965 Ford Mustang Shelby GT350 and the 2020 Ford Mustang Shelby GT350R represent different eras of automotive design and technology. The 1965 GT350 holds a special place in Mustang history as one of the early high-performance variants, while the 2020 GT350R is a contemporary track-focused machine that benefits from decades of technological advancements. Both models are iconic in their own right and appeal to enthusiasts with different preferences and interests.

domain: hobby


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THE Upper Hunter mining industry is a relatively low user of water flowing through the Hunter River system, according to new figures released by the Upper Hunter Mining Dialogue.
In 2016, the mining industry only used just two per cent of the water in the system according to figures collated by the dialogue’s water accounting project.
This compares to 36 per cent extracted from the river and used by non-mining businesses and residents.
The water accounting project’s results also show most of the water used by the mining industry comes from sources other than the river.
The mines sourced 55 per cent of all the water they used from onsite rainfall and runoff and a further 28 per cent was sourced from deep aquifers that are of limited used to other water users due to their high salinity.
Almost half of the water – 47 per cent - used in the mining sector was recycled and re-used.
The water accounting project was initiated through the dialogue to gain a better understanding of mining’s interaction with water resources in the region and identify opportunities for water management opportunities.
Dialogue chairman Dave O’Brien said the water accounting project’s annual findings were valuable tools to use in the dialogue’s work bringing together the mining industry with local residents, businesses, environmental groups and regulators to work together to minimise the cumulative impacts of mining.
“The water accounting project provides the Upper Hunter Mining Dialogue members and the community with information about mining’s interaction with water resources in the region, allowing for a more informed discussion,” he said.
“The project allows the mining industry to be a leader in the area of water management and provides the facts on water usage to the community.
“We understand water usage is something in which the community has a strong interest.
“The unique makeup of the Upper Hunter Mining Dialogue, which includes all active mining companies in the Upper Hunter, allows us to collect this water usage data which is very valuable, particularly in times such as the current drought.”
The water accounting project focuses on the Hunter River and its tributaries from Glenbawn Dam downstream to Singleton – where Upper Hunter mines and the major water supply dams are located – and collects the water usage data annually.
Upper Hunter mines get their water from a variety of sources and must be licenced for any water they take from rivers and groundwater.
According to the data released by the dialogue, only 10 per cent of the water used by mines came from rivers and alluvial aquifers.
As part of the Hunter River Salinity Trading Scheme, Upper Hunter mines are only allowed to discharge into the Hunter River at times when river flows are high.
Despite a wetter than average year in 2016 generating higher than average river flows, only four per cent of mine water was discharged into the river.

domain: news


A meeting on Thursday evening of area residents gathered for an event entitled “Save Birmingham from ALDOT’s plan for I-20/59 downtown” has launched an online effort to raise awareness about the current proposals being considered.
The group, calling themselves Rethink 20/59, has created a fan page on Facebook. They have also posted a link to a petition via Change.org directed at Birmingham mayor William Bell, the Birmingham City Council, the Jefferson County Commission and Alabama governor Robert Bentley asking them to “rethink the plan for Interstate 20/59” through the city center. The city and county had asked ALDOT last July to reconsider its initial plan to simply re-deck the existing section, leading to the current proposal. The council already stated during their June 18 general business meeting that they intended to meet with ALDOT’s director when he is in town next week.
A second petition launched on June 19 on Causes.com and created by Stuart Oates entitled Save Downtown Birmingham. It currently has 22 virtual signatures.
This latest development among those speaking out against plans to replace the decking between 31st St. N. and Malfunction Junction and redirecting local traffic patterns is not without precedent in the country. While efforts are underway in Dallas, TX to influence the removal of Interstate 345, the campaign most closely resembling Birmingham’s in the southeastern United States is in Louisville, KY, where 8664 is using Facebook in its fight to remove the Interstate and reconnect a waterfront park to the city’s downtown area. There is also a continuing effort in Syracuse, NY to look at alternatives to widening a stretch of I-81 through the heart of downtown (and coming extremely close to existing buildings in the process).
Photo: 2009 Rendering from ONB Breakfast Briefing presentation.


I’ve been watching a lot of anime these past few weeks. Some of it out of pure enjoyment, some out of the tense obligation I’ve created for myself to write a series in which I review three episodes of a new anime and decide whether I want to continue it or not. During this time, I’ve found myself jumping onto my favourite series as soon as an episode comes out, catching them once a week.
While other shows have been ones I’ve just watched back to back to back. Doing all of this, I’ve noticed that how I respond to an anime does seem to be impacted by how I consume it. In fact, there have been some shows I’ve really loved this year that I’m not sure I would have felt the same way about had I not binged a ton of it beforehand before catching up.
So it begs the question, is there a “better” way to watch anime?
Continue reading “Is there a best way to consume Anime? – Binge vs. Episodic”
I’d like to make the excuse that there wasn’t much going on this season due to lockdown and shows getting put on hiatus for a few months as a result. But if I’m being honest, there was a ton of anime out there to watch in reality, I just didn’t make the usual effort to go and try some different things like I had been doing the seasons previous.
That’s on me I guess. Then again, taking a breather for a month might not have been the worst thing. All I feel embarrassed about is how short this post is going to end up being as a result. But it’s all that Clone War’s fault.
Continue reading “2020 Spring Anime Season Roundup”
This post is probably going to be a bit briefer than the one I wrote for last season. Mostly because I’ve been a little better about actually following up on the seasonal anime series I’ve been watching here on my blog. Helped by the fact that I seem to have managed to find a bit of a groove again when it comes to a weekly upload schedule.
As we’re already starting the Spring season, and I’ve not watched anything yet except Digimon, I’ll just follow up all the shows mentioned in the Winter 3 Episode Rule series and link to any and all relevant articles I’ve already written on that show. Here’s a link to all of my impressions of the series I tried and which ones I kept on top of.
Continue reading “2020 Winter Anime Season Roundup”


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The Musical Fund Society actively invests in the musical life and legacy of Philadelphia by making limited funds available to incorporated, non-profit classical music organizations for projects which advance the foundational goals of the Society:
The Society does not fund the general operation of an organization, nor will it fund non-production related expenses such as receptions and social events. The Society funds only specific projects. Applying organization that sponsor multiple events within a given year, must identify the specific project for which funds are requested.
The Society will consider supporting projects in the Philadelphia area that focus on:
- new music, with an emphasis on Philadelphia-area composers and their works
- innovative programs
- development of new audiences
- music education (specifically, music programs; not scholarships)
- recording projects involving new compositions performed by local artists
While the aggregate funds available for distribution from the Society are modest, it is the hope of the Society that its grant will play a significant role in the funded project. Grants generally range from $2,000 to $6,000 per year and should be an important, but not the sole, source of funding. Applications that rely on the grant as the sole source of funding will be rejected.
Criteria for judging include, but are not limited to, the following:
- Artistic excellence
- The project’s relevance to the mission of the applicant organization
- The organization’s capacity to complete the project
Applications for multi-year projects will be considered. Grant applications should include a very specific chronology of events to occur during the multiple-year timeframe. An organization that receives a multiple-year grant should not expect further funding for the same project from the Society in the year following completion of the grant.
Applicant organizations whose staff or board includes current Society members are eligible to apply for and receive grants.
The Musical Fund Society requires that all grantees submit a final report after the completion of the funded project. Additionally, when posting about Society funded projects on social media, grantee organizations are expected to tag MFS social media pages in relevant posts. Consideration of future proposals is contingent upon compliance with these requirements.
Applications for FY 2024 grants must be submitted no later than 4:00 PM on March 1, 2023.

domain: finance


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This is a trifle different from our usual posts but poses some interesting questions. Is the Reformation really to blame for all our financial woes?
By Chris Bowlby BBC Radio 4.
Discussion among eurozone leaders about the future of their single currency has become an increasingly divisive affair. On the surface, religion has nothing to do with it – but could Protestant and Catholic leaders have deep-seated instincts that lead them to pull the eurozone in different directions, until it breaks?
Following the last European summit in Brussels there was much talk of defeat for Chancellor Merkel by what was described as a “new Latin Alliance” of Italy and Spain backed by France.
Many Germans protested that too much had been conceded by their government – and it might not be too far-fetched to see this as just the latest Protestant criticism of the Latin approach to matters monetary, which has deep roots in German culture, shaped by religious belief.
Churchgoing has been in decline in Germany as elsewhere as secularisation has spread, but religious ideas still shape the way Germans talk and think about money. The German word for debt – schuld – is the same as the word for “guilt” or “sin”.
The 19th Century Latin Currency Union struggled to survive after a number of countries minted and printed more money than they were meant to”
Talk of thrift and responsible budgeting comes instinctively to Angela Merkel, daughter of a Protestant pastor.
Merkel’s frequent assertion that “there is no alternative” to austerity policies (while reminiscent to Britons of Margaret Thatcher) has been likened to the famous stubborn statement by German Reformation leader Martin Luther: “Here I stand. I can do no other”.
The new German president, Joachim Gauck, who might play an important role in constitutional arguments about the single currency, is also from the Protestant fold – he is a former Lutheran pastor.
The country’s population is fairly evenly divided between Protestants and Catholics – as well as those of other faiths, or none – and Merkel’s and Gauck’s ascent symbolises changes in Germany since reunification in 1990.
Both lived in East Germany, a historically Protestant territory, while West Germany had several influential Catholic political leaders, who, in earlier post-war decades, had joined in broad Catholic enthusiasm for European integration.
Former West German Chancellor Konrad Adenauer, a Rhineland Catholic highly distrustful of Protestant Prussian traditions to the German east, led West Germany into the signing of the Treaty of Rome in 1957.
This created the European Economic Community, forerunner of today’s EU. And there was a clear geographical fit between the six countries which signed and the territory of Charlemagne’s Holy Roman Empire.
Charlemagne, claimed by modern European unifiers as a kind of patron saint, had created a new currency for his territories – the livre carolienne.
Helmut Kohl, who took Germany into economic and monetary union in the 1990s, was another Catholic Rhinelander constantly visiting cathedrals and speaking of the ancient spiritual roots of a united Europe.
There was much talk of the Germans sacrificing their beloved Deutsche Mark currency “on the altar of European unity”.
But German reunification at that time also meant the capital moving back to Berlin, away from closer Catholic connections felt in the west and south of the country.
And the eurozone crisis has intensified a deep-rooted debate about whether Germans, shaped by Protestantism, are fundamentally different from Catholic “Latin” countries and their allies.
German banking has, from medieval times, been more cautious than that in Italy and Spain. And sceptical Germans looking at the history of previous currency union troubles point to the 19th Century Latin Currency Union.
Germany, unifying under Prussian leadership through its own customs union, did not join. The Latin Union struggled to survive after a number of countries, notably the Papal States, minted and printed more money than they were meant to.
Politicians or states undermining money have diabolical overtones for angst-filled Germans.
In Goethe’s Faust, one of the most famous works in German culture, Mephistopheles persuades the Holy Roman Emperor to issue a new paper currency – despite one of his advisers warning that this is the counsel of Satan.
Order duly breaks down as the Emperors’ subjects go on a binge bearing no relation to their real wealth.
Weimar Republic hyperinflation in the early 1920s – when “money went mad” and all moral as well as economic order was seen as collapsing – seemed a diabolical vision made real.
Some in Germany suggest today’s eurozone would be better dividing, with some kind of Latin Union on one side, and on the other a German-led group of like-minded countries including perhaps the (Calvinist) Dutch and the (Lutheran) Finns.
The former head of the German industry association, Hans-Olaf Henkel, has said that “the euro is dividing Europe”.
He wants the Germans, Dutch and Finns to “seize the initiative and leave the euro”, creating a separate northern euro.
A new split along ancient lines? The government in Berlin has begun to plan for what it sees as a hugely significant anniversary in 2017 – 500 years since Luther began The Reformation.
He was protesting against indulgences, a controversial attempt by the Papacy to solve its fiscal problems by persuading Europeans to buy absolution from their sins.
One German commentator, Stephan Richter, has suggested mischievously that the eurozone’s problems would have been prevented if only Luther had been one of the negotiators of the Maastricht treaty, deciding which countries could join the euro.
‘Read my lips: No unreformed Catholic countries,’ he would have chanted. The euro, as a result, would have been far more cohesive,” says Richter.
Richter is himself a Catholic, but an admirer of thrifty economics. “Too much Catholicism” he suggests, “is detrimental to a nation’s fiscal health, even today in the 21st Century”.
But he believes some historically Catholic countries, such as Austria and Poland, may have come under more Germanic influence due to their geographical proximity. They are “Catholics perhaps, but with a healthy dose of fiscal Protestantism,” he reasons.
Commemorations in 2017 will doubtless try to stress that Reformation divisions between Protestants and other reformers and Catholicism were not too great.
But the usually thrifty government under Chancellor Merkel has already promised 35 million euros to mark this birth of Protestantism.
And where will the eurozone be in 2017?
Still intact? Or coming to terms with a new historic divide between the Latins and the preachers of Protestant thrift?
Chris Bowlby presents Analysis on BBC Radio 4 and has reported extensively on the eurozone crisis.


Bribery, Corruption, Money Laundering: The Many Investigations Into Trump Business Partners
WASHINGTON — Hary Tanoesoedibjo, a wealthy Indonesian financier who had are associated with President Donald Trump to build two indulgence useds in Bali and who attended Trump’s inauguration in January, was banned from leaving his country on Monday after becoming the subject of a national police investigation.
Tanoesoedibjo is accused of menacing an researcher in Indonesia’s attorney general’s office in a series of text words cast in 2016.
The Tanoesoedibjo investigation is just the latest of countless concerning both current and former Trump business partners for felonies such as bribery, corrupt practices and, most often, coin laundering.
In India, Trump has partnered with two different corporations that are currently or have recently been under investigation by local authorities. According to The Washington Post, IREO, the Indian investment firm that Trump has licensed his appoint to for a tower in the city of Gurgaon, was under investigation for alleged fund laundering and illegal tract purchases. M3M, another Indian house licensing Trump’s name for a different fortres in Gurgaon, has regularly been targeted for failing to pay taxes and now faces a criminal complaint alleging bribery. The head of IREO, Lalit Goyal, told The Washington Post that his firm is no longer under investigation.
In December, after Trump won the U.S. presidential election but before he officially announced he would retain possession of the Trump Organization, he canceled copes to license his name to real estate enterprises in Azerbaijan, Brazil and Georgia. There were concerns about corrupt practices and money laundering in the Azerbaijan and Brazil lots, though apparently none directly related to the Georgia deal.
A hotel and an office tower complex that were supposed to bear Trump’s name in Rio de Janeiro have been under inquiry for the suspect action of pension fund managers whose stores backed the assets and other real estate. The funds formally grew under investigation by the Brazilian federal prosecutor’s office in October 2016 as part of a broad-minded dishonesty investigation.
The Brazilian sleuths are also looking into whether the Trump Organization advantaged in any way from dishonesty or bribery, according to Bloomberg, although the president’s corporation itself is not an objective of police investigations. Trump canceled the batch for the Rio de Janeiro projections in December 2016. The Trump Organization has now been claimed it was only licensing the Trump name to the buildings and had no other involvement in the investments.
The Trump Organization wasted a lot of age strengthening the future Trump Tower in Baku, Azerbaijan before canceling the deal in December 2016. The deal involved licensing the president’s word to an Azerbaijani development company run by Anar Mammadov, the lad of that country’s director. A 2017 report in the New Yorker revealed notions that the castle benefited from financing that appeared to be part of an Iranian Revolutionary Guard money laundering arrangement. Mammadov has not publicly commented on the deal.
The treats in Azerbaijan and Brazil apparently exclusively became groundless formerly Trump faced the added inquiry that comes with being the president of the United States.
But Trump is no stranger to the same kind of investigations, litigations and accusations of money laundering or other evil that his former and current business partners front. Trump’s businesses have paid vast fines and colonizations for fraud and coin laundering, although he has rarely admitted regret.
The Trump Organization did not respond to a request for comment on the investigations.
Trump paid $477,000 to the Treasury Department as part of a 1998 licensing agreement for the Trump Taj Mahal casino’s 106 violations of anti-money cleaning laws. In 2015, Trump Taj Mahal paid a $10 million fine to the Treasury Department’s Financial Crimes Enforcement Network for further violations of anti-money laundering constitutions. Trump no longer owned the casino at that time, but the settlement stated that irreverences extended back to 2003, when he was the owner.
In 1991, Trump was also forced to pay a rather small-time punishment of $30,000 after his father, Fred Trump, purchased $3.5 million merit of chips at the Trump Castle casino( now under new possession with the name Golden Nugget Atlantic City) in Atlantic City to help prevent it from testifying bankruptcy. New Jersey casino regulators determined this was an illegal loan.
Then there is Trump SoHo, the inn and condo building in downtown Manhattan that the Trump Organization and the Bayrock Group, an investment firm, completed in 2008. Bayrock Group conduct included Felix Sater, a former FBI informant with ties to Russian and American organized crime who controlled the Trump SoHo deal, and Tevfik Arif, who was arrested in Turkey in 2010 for allegedly leading a prostitution resound( those fees were finally lowered ).
Trump too partnered with Bayrock Group on a condo asset in Fort Lauderdale, Florida, which eventually went into default after Trump attracted his mention from it. He also demonstrated Bayrock a temporary, one-year exclusive right to create a Trump Tower in Moscow that was never built.
Bayrock Group’s ex-finance chairman, Jody Kriss, has filed numerous disputes against his former bos, to suggest that the company and the Trump Soho project were conduits for fund laundering. Separate court filings in the U.S. and Switzerland allege that Kazakh billionaire Viktor Khrapunov and his son, Ilyas Khrapunov, laundered illicit monies stolen from a Kazakh city’s treasury through comfort condo purchases at Trump SoHo and the Fort Lauderdale Trump property. The Khrapunovs claim the charges brought by the Kazakhstan government are politically motivated.
A number of countries where the Trump Organization conducts business grade high on the Corruption Perception Index bring out by the international anti-corruption radical Transparency International, replied Shruti Shah, a due diligence professional at the anti-corruption nonprofit Coalition for Integrity.
Shah told HuffPost that these distributes were” troubling not just because of the CPI score of the countries mentioned, but too because, in a number of cases like Azerbaijan, the business partners are closely associated with government officials .”
The involvement of foreign government officials in the Azerbaijan deal is noteworthy because the Foreign Corrupt Practices Act precludes U.S. conglomerates from supplying is favourable to a foreign government official as part of a business deal, even if they claim the latter are unaware they stipulated such a benefit.
” Doing suitable due-diligence would have been able to divulged these issues ,” Shah said.
Since taking office, the Trump Organization has hired a compliance officer and attempted to apply greater scrutiny to its treats. This greater level of due diligence played a role in the company’s decision to turn down a partnership with Mukemmel Sarimsakci, a Dallas-based developer known as the” Turkish Trump ,” to license the Trump Organization’s new Scion brand name to hotels in Dallas and St. Louis.
Read more: http :// www.huffingtonpost.com /~ ATAGEND


text: The Queen of Pieterburen Bed & Breakfast is a place with artistic atmosphere, where children and adults may enjoy a holiday to remember. Occupying a historic house dating back to 1870, this lovely B&B in Pieterburen impresses with its fairy-tale-like atmosphere. The Queen of Pieterburen B&B has an enchanting garden with lots of cosy nooks and patio as well as two beautifully decorated, one of which is for 5 guests and has a private shower room. Toilet is in the hallway. Guests may enjoy creative shops with painting, decorating mirrors, making jewelry, etc. Pieterburen and the area are also perfect for canoeing, walking, horse riding, sailing, birdwatching.


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Free U.S shipping on orders over $50
Original Illustration on paper by Zach Welch. It features 80's themed colors with bold brush strokes.
Size: 6 x 6 inches
Media: Copic markers & India ink on paper
This work is unframed.
Free Shipping on U.S orders over $50
and International orders over $150.
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All orders ship from Frisco, Tx.

domain: artistic


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No School Is an Island—Except on the Coast of Maine
No school should be an island, isolated from the world around it. But, off the coast of Maine, a small number of year-round island communities, reachable only by boat, strive to preserve their schools as the soul of their communities. Without the school, there is no island community. Ranging in size from a handful to 220 students, these island schools face a unique set of environmental, cultural, and curricular challenges that are representative of isolated rural schools throughout the country.

domain: geography


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Entering Myanmar's Market with Meidensha
Left to right: Takehiro Honda, CRO of ASEAN and Oceania; Kozo Masaki, executive vice president of Meidensha; Nuan Yun Oo, director of Yangon Bureau, Ministry of Industry of Myanmar; Win Aung, president, the Republic of the Union of Myanmar Federation of Chambers of Commerce & Industry; and Chit Khaing, chairman of AGE
Metal One, Meidensha Corporation, and Asia General Electric Co., Ltd. (AGE)—Myanmar's largest manufacturer of transformers for electric power companies—signed a memorandum of understanding in December last year regarding plans of Metal One and Meidensha for investments and board participation in AGE.
Democratization is proceeding in Myanmar, and increasing demand for transformer substations is anticipated as the country prepares to meet the expected robust demand for electric power. The transformer substations currently installed are obsolete, and the resulting electric power losses have become a major obstacle to economic growth. There is an urgent need to build transmission and distribution grids.
AGE will manufacture the latest types of transformers, in sizes small to large, through a technical tie-up with Meidensha, which will also help to renovate or replace aging facilities. Meidensha will gain its second Southeast Asian manufacturing and retailing site to supplement the one it has in Singapore. Metal One will increase its supplies of grain-oriented electrical steel sheet and other types of sheet for transformers, making it more of a force in Myanmar's steel market.
(Products for Electrical Industries & Steel Sheet Department)


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Student Video Awards’ one-day workshop
9:00 am - 5:00 pm
151 West Cordova Street
Learn from some of the sharpest minds in video production and documentary filmmaking from Vancouver at the Student Video Awards’ one-day workshop.
This is an opportunity to discover the latest techniques in a range of topics from the art of visual storytelling and cultivating creative ideas on how to make your video go viral as well as tips and tricks from top animators.
The workshop is part of the third annual Vancouver Sun Student Video Awards, which offers more than $20,000 in prizing to Vancouver Film School’s Summer Intensives series. Since the inception of the SVA in 2011, more than 1,500 students have participated in submitting films which have reached more than 80,000 people. For more details, go to vancouversun.com/studentvideos.
Cost: $50 – includes lunch and a workshop t-shirt
Speakers – Morning sessions
– Art of telling a story – Duncan McCue, reporter, CBC News – The National
– Putting the I in independent – Mark Yuen, Vancouver Sun award-winning videographer
– Push the Creative Envelope – David Vandas, creative entrepreneur, video director
– Online Distribution Strategies – Kai Nagata, journalist, videographer
– The Working Artist – Vasho Pekar, filmmaker, VFX artist
– Finding Your Creative Mix – Corwin Hiebert, creative consultant
Speakers – Afternoon sessions
– Storytelling and Character Development – Michael Baser, writer/producer and Head of Writing for Film & Television at VFS
– The Age of Imagination-focusing on animation & idea generation – Colin Giles, Senior Instructor in 3D Animation & Visual Effects department at VFS
– Mixing Media – Nida Khan, freelance motion designer, VFS Digital Design instructor
– Animation Tips and Tricks – Heather Garrow, animator, VFS 3D Animation & Visual Effects instructor
– Genres in Cinema – Paul Jensen, walking film encyclopedia, VFS Writing for Film & Television instructor
– Moving Camera Demonstration – Ted Jones, film production veteran, Head of Film Production at VFS


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Professor Don Grubin, who is running a pilot on lie detector tests for sex offenders on behalf of the Ministry of Justice, thinks the drastic move could work on a voluntary basis.
The academic, a professor of forensic psychiatry at Newcastle University's Institute of Neuroscience, is already running a Government-backed programme that offers so-called "chemical castration" for sex offenders where medication reduces an individual's testosterone levels.
But following a visit to the Czech Republic, where they offer physical castration for volunteers, Prof Grubin believes there is scope to go even further in Britain.
Asked by More4 News whether there is a place for physical castration in Britain, he said: "If you asked me that two years ago I would have said no.
"Having thought a bit about what they say is going on here (Czech Republic), and the way they use it here, I'm coming to the view that there may be a small number of offenders in Britain who would benefit from physical castration offenders who have had medical treatments that have shown it's helpful for them, and who consent to the idea that this would be very helpful to them."
He said there are a small number of sex offenders who do request such a move.
But Juliet Lyon, director of the Prison Reform Trust, said such as move would be a "step too far in a justice system committed to humane, decent and effective treatment of offenders".
"To reduce risk of re-offending, surely we should build on the proven success of sex offender treatment programmes in prison, intensive probation supervision in the community and 'circles of support' work pioneered by the Quakers," she added.
Authorities in the Czech Republic offer physical castration to those sex offenders who request it.
In tonight's programme, one serial offender referred to only as Vaclav, who assaulted girls as young as 12, said it was the "best decision" he has ever made.
He adds: "On the one hand you have to protect the potential victims and on the other hand I wanted to be protected from myself, I wanted to live like a normal person."
Prof Grubin is currently running a three year programme that offers drugs to sex offenders to reduce the risk of further offending.
The programme involves jailed sex offenders volunteering to take medication to reduce their testosterone levels to those of prepubescent boys. The result is similar to the effects of castration.
He is also heading up a Government pilot programme to force sex offenders in the Midlands to take lie detector tests when they are released from prison.
:: The interview is on More4 News from 8pm tonight.


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Lime N Lemon Embroidered Handheld Tote
Regular price $62.00
Shipping calculated at checkout.
Colorful Summer Bag with embroidered Lime and Lemon pattern. Inside pockets. Fun Tassel. Everything to brighten your outfit.
Come with a strap for a cross body use.
90% Jute, 10% Lurex
12"H x 15W" x 3"D

domain: design