We found that minor hypoxia (10% oxygen) significantly increased the migration of vascular endothelial cells while hypoxia (8% oxygen) significantly inhibited cell migration. The present study was performed using cells cultured in 10% oxygen. RNA interference was used to block the endogenous generation of hydrogen sulfide by CSE, CBS, or MPST in a vascular endothelial cell migration model in both normoxia and hypoxia. The results showed that CBS had a promoting effect on the migration of vascular endothelial cells cultured in both normoxic and hypoxic conditions. In contrast, CSE had an inhibitory effect on cell migration. MPST had a promoting effect on the migration of vascular endothelial cells cultured in hypoxia; however, it had no effect on the cells cultured in normoxia. Importantly, it was found that the hypoxia-induced increase in vascular endothelial cell migration was mediated by MPST, but not CSE or CBS. The western blot analyses showed that hypoxia significantly increased MPST protein levels, decreased CSE protein levels and did not change CBS levels, suggesting that these three hydrogen sulfide-producing systems respond differently to hypoxic conditions. Interestingly, MPST protein levels were elevated by hypoxia in a bi-phasic manner and MPST mRNA levels increased later than the first stage elevation of the protein levels, implying that the expression of MPST induced by hypoxia was also regulated at a post-transcriptional level. RNA pull-down assay showed that some candidate RNA binding proteins, such as nucleolin and Annexin A2, were dissociated from the 3′-UTR of MPST mRNA in hypoxia which implied their involvement in MPST mRNA regulation.Hypoxia-induced angiogenesis is a common phenomenon in many physiological and patho-physiological processes. However, the potential differential roles of three hydrogen sulfide producing systems cystathionine γ-lyase (CSE)/H Angiogenesis, a process that mainly includes the proliferation, migration, and tube formation of vascular endothelial cells, is a common phenomenon in many physiological and patho-physiological processes, including organ development, wound healing, tumor development, and metastasis  significantly promoted collateral vessel growth and increased regional blood flow in ischemic tissue in a rat model of critical limb ischemia , cystathionine β-synthetase (CBS), and 3-mercaptopyruvate sulfurtransferase (MPST) with L-cysteine and homocysteine as their substrates  and culture medium  were purchased from Allcells . Cells were cultured in gelatin-coated dishes at 37°C in an incubator containing 21% OConfluent HUVECs were starved overnight and were scratched using a pipette tip for the wound healing assay. Markings were drawn on the culture dishes as reference points so as to make sure that the same visual field was photographed at 0 and 24 h. Typically, 8–12 visual fields were chosen randomly in one culture dish. Cells were photographed using an EVOS fl Microscope  after incubation in normoxic or hypoxic conditions. The outline of the wound area (the area with no cells) was then drawn using Image J software to get the exact pixel coverage of each wound area. Then, the pixel coverage value of each wound area was converted to a standard value, shown as one data point, by being divided by a fixed value so as to normalize the average value of the control group to be 100.siRNAs specific for human CSE, CBS, or MPST are as follows: human CSE siRNA: sense 5′-CUAUGUAUUCUGCAACAAATT-3′, antisense 5′-UUUGUUGCAGAAUACAUAGAA-3′; human CBS siRNA: sense 5′-CUCACAUCCUAGACCAGUATT-3′, antisense 5′-UACUGGUCUAGGAUGUGAGAA-3′; human MPST siRNA: sense 5′-GGAAUUCCGUUGACUUGUUTT-3′, antisense 5′-AACAAGUCAACGGAAUUCCTG-3′.siRNA specific for human CSE, CBS, MPST, and scrambled siRNA as a negative control were purchased from Thermo Scientific-Ambion. HUVECs were transfected using Lipofectamine RNAiMAX (Thermo Scientific-Invitrogen) and OPTI-MEM (Thermo Scientific-Gibco) according to the instructions provided by the manufacturer and then the cells were cultured for another 48 h for further analyses.Confluent HUVECs were starved overnight and were cultured in normoxic or hypoxic conditions. Then, cells were lysed in a 1 × sodium dodecyl sulfate (SDS) sample buffer . Bicinchoninic acid (BCA) reagent was purchased from Shen Neng Bo Cai Corp.  and the BCA method was used for protein concentration determination. Protein samples (50 μg) were subjected to electrophoresis in a 10% SDS-polyacrylamide gel, and then transferred to a polyvinyl difluoride membrane . After blocking in 5% non-fat milk in Tris Buffered Saline with Tween-20  for 2 h at room temperature (RT), the membrane was incubated with primary antibodies at 4°C overnight. Anti-CBS and anti-β-actin were purchased from Proteintech Group . Anti-CSE and anti-MPST were purchased from Santa Cruz Biotechnology . After washing the membranes using TBST and then incubating them with horseradish peroxidase-conjugated secondary antibodies for 2 h at RT, SuperSignal West Pico Chemiluminescent Substrate  was used for band detection. Band signals were then quantified using Smart viewer software.Total RNA was extracted from cells using Trizol reagent. RNA was reverse-transcribed using a cDNA synthesis kit . Real-Time PCR was performed using a StepOnePlus Real-Time PCR Detection System . A total volume of 20 μL reaction mixture containing 1 μL cDNA, 10 μL SYBR Green PCR Master Mix  and 1.6 μL each primer (10 μM) was used for this assay. GAPDH was used as reference for normalization and the relative expression of mRNA was calculated according to the ΔΔCt method.The primer pairs used for the Real-time PCR were as follows: for the human MPST gene, the forward primer was 5′-CGGAGTCTCCTCCCTTTGGT-3′, and the reverse primer was 5′-CCTCCCTAAGATGCAGCTCG-3′. For the GAPDH gene, the forward primer was 5′-TGCCCCCATGTTCGTCA-3′, and the reverse primer was 5′-CTTGGCCAGGGGTGCTAA-3′.The human MPST mRNA 5′-UTR and 3′-UTR were amplified from HUVECs using the following primers: 3′-UTR, the forward primer was 5′-AGCTGGGCAGGACACAGG-3′, and the reverse primer was 5′-TTCCTCAAAAATAAAACAGAAAGGAGG-3′. 5′-UTR-exon1, the forward primer was 5′-GGAAGGCGCGGGCAGCAGCGGCTCCGAGTGGCCGCGGCGGTGGGCTGTGCC-3′, the reverse primer was 5′-ATTGGCGACCTGCAGCGGACCAAAGGGAGGAGACTCCGGCACAGCCCACCGCC-3′. 5′-UTR-exon2, the forward primer was 5′-CGCTGCAGGTCGCCAATATAAATGCTTGATGAG-3′, the reverse primer was 5′-CTGCGGGCCTCCACTTGCTGGGCAGG-3′. 5′-UTR-exon3, the forward primer was 5′-AAGTGGAGGCCCGCAGCCCGAGTGTCGCCGCC-3′, the reverse primer was 5′-GGCGGCGACACTCGGGCTGCGGGCCTCCACTT-3′. The 3′-UTR or 5′-UTR  was subcloned into the pGEM-T vector.2) to form the secondary structure.RNA transcripts were produced by T7 RNA polymerase  with Biotin RNA labeling Mix  in an 18 μl system. After incubation for 2 h at 37°C, 2 μL RNase-free DNase was added and incubated for 15 min at 37°C to remove template DNA. Then, 2 μL 0.2 M EDTA (pH = 8.0) was added to stop the reaction. Biotinylated RNA was added into the Structure Buffer  according to the instructions provided by the manufacturer. Biotin-labeled antisense RNA was used as a control in all experiments. Magnetic beads were washed three times in 500 μL Wash Buffer I  and resuspended in Wash Buffer I. Biotinylated RNA was incubated with magnetic beads at 4°C overnight. Biotinylated RNA-streptavidin magnetic beads were incubated with 1 mg cell extract with RNase inhibitor at RT for 1 h. The RNA/protein/bead complexes were washed three times using Wash Buffer II . Then, proteins were eluted by boiling in 5 × SDS PAGE loading buffer and resolved using SDS-PAGE. Protein bands were silver-stained and cut for mass-spectrometric analysis .3Fe(CN)6 and 50 mM NaS2O3] at RT for 5 min. The gel pieces were dried in 50% acetonitrile (ACN) at RT for 30 min and then dried in 100% ACN at RT for 30 min. Protein was digested using 0.4 μg sequencing grade trypsin at 37°C for 16 h. The digested peptides were extracted from the gel pieces using Extraction Solution (water solution containing 5% trifluoroacetic acid and 67% ACN) at 37°C for 30 min and then applied to the on-line liquid chromatography (LC)-collision-induced dissociation (CID)-MS-MS analysis.Protein bands from SDS-PAGE gels were excised. The gel fragments were cut into small pieces and decolored in 50 μL decoloring solution [15 mM K+~5+ charges). Total cycle time was set to 2.5 s. For CID, a rolling collision energy setting was applied to precursor ions. Proteins were identified using the MASCOT V2.3 search engine . Search parameters were as follows: two missed cleavage sites; fixed modifications of Carbamidomethyl (C); partial modifications of Acetyl (Protein N-term), Deamidated (NQ), Dioxidation (W), Oxidation (M); precursor ion tolerance is ± 30 ppm, fragment ion tolerance is ± 0.15 Da.High-performance liquid chromatography was carried out to isolate trypsin-digested protein fragments on a 75 μm inner diameter 15 cm reverse-phase capillary column, 3.0 μm, 120 Å, C18 (ChromXP Eksigent) using a gradient from 5 to 35% buffer B  over a period of 90 min. Mass spectrometry was performed on a TripleTOF5600 system (AB SCIEX) using electrospray ionization. The optimum conditions: ion spray voltage, 2.5 kV; curtain gas, 30 psi; nebulizing gas (GS1), 5 psi; interface heater temperature, 150°C. For information dependent acquisition, survey scans were acquired in 250 ms. At most, 35 product ion scans were collected exceeding a threshold of 150 counts/s . Statistical analyses were performed using GraphPad Prism statistical software 7.0 . The Shapiro–Wilk normality test or the Kolmogorov–Smirnov normality test was used. Data were analyzed using unpaired After starving overnight, HUVECs were scraped using a pipette tip for the wound healing assay. Then, the cells were cultured in normoxic or hypoxic conditions with 8 or 10% oxygen levels. Results showed that hypoxia (10% oxygen level) significantly increased cell migration compared with cells cultured in normoxia Figures . HoweverCSE protein levels were significantly decreased using RNA interference Figures . HUVECs CBS protein levels were significantly decreased using RNA interference Figures . HUVECs MPST protein levels were significantly decreased using RNA interference Figures . HUVECs HUVECs in hypoxia migrated faster than those in normoxia after transfection with NC siRNA . Therefore, the cells were cultured in hypoxia for the times indicated in Figure  h. ThereHUVECs starved overnight were treated using Trizol reagent for further real-time PCR analyses. Results showed that MPST mRNA levels were not increased at 4 h in hypoxia when MPST protein levels had already been elevated Figure . These rTo investigate the post-transcriptional regulation of MPST mRNA by hypoxia, the 5′UTR and 3′UTR of MPST mRNA with biotin tags were prepared. RNA pull-down experiments were performed by incubating biotin-tagged mRNA with lysates of HUVECs after culturing in normoxic or hypoxic conditions. The antisense sequence of the UTR region was used as a negative control. The mixture was then incubated with streptavidin-coated beads to obtain biotin-tagged RNA with binding proteins on it. SDS-PAGE with silver staining showed that many proteins bound with the 5′UTR, rather than the 3′UTR of MPST mRNA in normoxic conditions compared with their antisense RNA groups Figure . HoweverIn order to identify the binding proteins of MPST mRNA which were significantly decreased at the protein level or dissociated from MPST mRNA, the protein bands indicated with arrows and numbers in Figure in Vitro and in Vivo (Cai et al., 2S in ischemic disease models (Wang et al., 2S in angiogenesis have been investigated. It has been reported by Cai et al. that the PI3K/Akt pathway was involved in its proangiogenic effect, while VEGF elevation was not observed in vascular endothelial cells after H2S treatment (Cai et al., 2S, indicating that the proangiogenic effect of H2S might involve cell-cell interactions between vascular endothelial cells and skeletal muscle cells (Wang et al., 2S in astrocytes. In accordance with that, VEGF and Ang-1 in the ischemic brain were also elevated by H2S. Moreover, ERK and HIF-1 (Jang et al., ATP channel and p38 (Papapetropoulos et al., 2S.It has been previously reported that hydrogen sulfide has a significant promoting effect on the process of angiogenesis both 2S have been reported, one of the most challenging questions in this field is the identification of “receptors,” namely the direct target of H2S. We reported previously that H2S could directly increase the activity of recombinant VEGFR2 in a cell-free system and found that there was a new disulfide bond C1045-C1024 in the kinase domain of VEGFR2 which could be broken by H2S, suggesting that VEGFR2 might be a “receptor” for H2S (Tao et al., Although, numerous signaling molecules involved in the effects of H2S, CBS/H2S and MPST/H2S. We have previously found that CSE and CBS were located differently in vascular endothelial cells (Tao et al., 2S produced from these enzymes could not reach target proteins far from the enzyme, since H2S could be easily oxidized (Cuevasanta et al., 2S would be too low to have any effect. In the current paper, it was found that CSE, CBS and MPST were playing different roles in the migration of vascular endothelial cells. As shown in Figures 2S was playing an inhibitory role in vascular cell migration. As shown in Figures 2S was probably playing a promoting role in vascular cell migration. As shown in Figures 2S was playing a promoting role in vascular cell migration only in hypoxic conditions. These findings indicate for the first time that CSE, CBS and MPST play different roles in vascular cell migration, although they all produced H2S. It has been reported that the distinctive expression pattern of CSE and CBS is closely related to phenotype in human mesenchymal stromal cells. CSE up-regulation was restricted to those cells undergoing mineralization, while CBS, not CSE expression levels were significantly increased during chondrogenesis (Gambari et al., There are three endogenous hydrogen sulfide producing systems in mammalian cells, CSE/H2S produced from MPST in mitochondria could decrease ROS levels that mediate hypoxia-induced endothelial cell migration. However, why MPST/H2S, but not CSE/H2S or CBS/H2S, mediated hypoxia-induced vascular endothelial cell migration and whether there are different specific direct target proteins, or “receptors,” for different H2S producing systems, will need to be further investigated.In contrast with CSE and CBS, MPST localizes in mitochondria as well as in the cytosol (Nagahara et al., It was found that the increase in MPST protein levels appeared earlier than the increase in MPST mRNA levels, suggesting that MPST might also be regulated at a post-transcriptional level. RNA pull-down experiments showed that there might be multiple RNA binding proteins on the 5′UTR of MPST mRNA, rather than the 3′UTR. Moreover, these binding proteins “disappeared” when vascular endothelial cells were cultured in hypoxia. Therefore, we speculate that those binding proteins might have a role in regulating MPST mRNA stability or translation and they might be regulated by hypoxia. Among those candidate binding proteins listed in the Supplementary Tables, some have been reported previously to be RNA binding proteins, such as nucleolin (Fahling et al., Figures In the current study, it was found for the first time that CSE, CBS, and MPST played different roles in the migration of vascular endothelial cells in normoxia and hypoxia. In addition, MPST, not CSE or CBS mediated hypoxia induced cell migration, probably through a post-transcriptional mechanism.BT and YZ conceived and designed the experiment. BT, RW, and CS performed the experiments. BT analyzed the data. BT and YZ wrote the paper. All authors have contributed to the paper and approve the submission of the manuscript.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.


The aim of this study was to investigate prevalence and related factors of androgen receptor (AR) expression in Thai breast cancer patients. A descriptive study was done in 95 patients, who were admitted to Charoenkrung Pracharak Hospital, Bangkok (2011–2013). Statistical relationships were examined between AR protein expression, tumor status, and patient characteristics. Compared with those from Western countries, ethnic Thai patients were younger at age of diagnosis and had a higher proliferative index (high Ki-67 expression), which indicates unfavorable prognosis. In addition, 91% of the Thai breast tumors that were positive for any of the following receptors, estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) also expressed the AR protein, while in triple negative breast tumors only 33% were AR positive. ER and PR expression was positively related with AR expression, while AR expression was inversely correlated to Ki-67 expression. AR status was strongly correlated with ER and PR status in Thai patients. There is an inverse relationship between Ki-67 and AR, which suggests that AR may be a prognostic factor for breast cancer. Breast cancer is by far the most common type of cancer among women around the world. In 2012, there were about 1,671,000 new cases of female breast cancer worldwide . InteresThere is scientific consensus that breast cancer is a systemic disease and multimodality treatment is needed to cure or prevent residual cancer after surgery ,5. The tMortality and morbidity are higher for patients with triple negative breast cancer, which has a negative ontology for hormonal receptors ,12. At pThere is growing evidence that the androgen receptor (AR) might be a new target for systemic breast cancer treatment ,16. SeveThis exploratory study enrolled a total of 95 Thai breast cancer patients that were admitted in the Charoenkrung Pracharuk Hospital  between October 2011 and October 2013. Clinical and pathological data were collected from the patients’ records. These data included age, menstrual status, body mass index (BMI), pre-operative pathology, previous diagnostic method, tumor size, tumor location, clinical nodal status, type of surgery, tumor stage, final pathology, pathological nodal metastasis, nuclear grade, margin of lymphovascular invasion (LVI), neural invasion, proliferative index (Ki-67), status of the ER, progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) responsiveness. The immunohistochemistry staining process was done with a Bench Mark GX automatic machine . The level of receptor expression was reported by a Thai board qualified pathologist. Rabbit monoclonal primary antibodies (clone SP1), rabbit monoclonal primary antibodies (clone 1E2), anti-HER2 (4B5) rabbit monoclonal primary antibodies, and rabbit monoclonal primary antibodies (clone 30-9)  were used to stain ER, PR, HER-2 and Ki-67, respectively.All patients who enrolled in the study gave a written informed consent. In addition, these patients received full information and explanation about the study objectives and rights that the patient has before enrolling in the study. The study was approved by the Ethics Committee for Researches Involving Human Subjects at the Bangkok Metropolitan Administration , on 21 October 2011, with the project identification number 073.54. This research has been carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki).+ and AR− breast tumor tissue from Thai patients in this study.The presence of the androgen receptor protein was tested in the remaining pathological specimens of the breast tumor tissue by using an anti-androgen receptor (SP107) rabbit monoclonal primary antibody , which was stained with the immunoperoxidase method . The immz = 1.96 at alpha 0.05% or 95% confidence intervalp = Proportion of androgen receptor positive; 70%, according to Ren et al. [n et al. d = Acceptable error of 10% = 0.01n = (1.96 )2 · (0.7) · (1−0.7)/(0.1) · 2 = 81 casesTo achieve our primary research objective, the following sample size formula for single proportion was applied:This calculation indicated that at least 81 patients had to enroll in this study in order to achieve enough statistical power.2 test was used for categorical variables and Student’s t test was used for numerical variables with normal distribution. A p value <0.05 was considered statistically significant. Logistic binary regression was used for the multivariate analysis.The statistical analysis of the clinical and pathological data was done with descriptive and inferential statistics. For descriptive analysis, the total values and percentages were used as categorical variables and the mean value with standard error of the mean for the numerical values. In addition, a comparative analysis was carried out using bivariate and multivariate analysis; the χA total of 95 ethnic Thai breast cancer patients were enrolled in this study. Their mean age was 51.37 ± 1.17 (mean ± standard error). Mean body surface area (BSA) and BMI were 1.56 ± 0.01 and 24.38 ± 0.46, respectively. Forty-one cases (43.16%) were in natural post-menopausal state. Axillary nodes were clinically palpable in twelve cases (12.63%), while 46 cases (48%) were pathologically node positive.Invasive ductal carcinoma was the most common diagnosis in these patients (95.79%) and about 56% of the cases had lympho-vascular invasion. About 70% of the patients had a high proliferative index (Ki-67 expressed ≥20%). The pathological staging at the time of treatment was equally distributed with about 30% of the patients in a late stage of disease (Stage 3).Approximately 60% of the malignant breast tumors were ER and PR positive, 40% had HER-2 receptor expression (HER-2 positive 2+ and 3+), while only 19% were of triple negative ontology. Of all tumors, 80% (76/95) expressed the AR protein. For more detailed information, see p value <0.001)  of the triple negative tumors showed an expression of the AR. The factors  related to AR expression were statistically analyzed. The expression of the AR was strongly related to ER expression. In addition, it was found that a triple negative tumor status was inversely related to the expression of the AR ( <0.001) .p < 0.001, 0.014, and 0.006, respectively). However, in the triple negative tumors, the H-score of the AR was approximately threefold lower than in the hormonal positive tumors (p < 0.001). These quantitative differences in the H-score of AR expression with expression of other  receptors are shown in + and AR− tumors with the cell proliferation marker Ki-67 was also analyzed, and it was found that AR− tumors had an approximate 50% higher Ki-67 expression than AR+ tumors (p < 0.01)  .This study is the first to describe AR protein expression in Thai breast cancer patients. Demographic data of Thai breast cancer patients are clearly different from those of Western patients. Unlike those from Western countries, breast cancer patients in Thailand generally come to the hospital with a more advanced, late stage of the disease. This is illustrated by our data in which our patients had a higher breast cancer stage than that generally observed in studies from Western countries. In fact, 15 cases in our study needed chemotherapy before surgery , representing 16% of the patients. The incidence of neoadjuvant chemotherapy in Western countries is usually less than 10% ,25. In o+ tumors regardless of ER status, since it can block both AR- and ER-mediated tumor growth. Clearly, AR and ER status of a breast tumor will determine the effect of an (anti)androgen on breast tumor cell proliferation.There is emerging evidence that the androgen receptor might be an additional target for systemic breast cancer treatment ,16; howeThe AR expression in our Thai patients was about 80%, which is comparable to previous reports from Western countries ,37. Noti+ breast cancer patients had a better survival prognosis than those who were AR− [Another interesting finding in our study was the fact that the expression of the AR was inversely correlated with the non-ER-dependent proliferation index Ki-67. This is an essential protein for progression through the cell cycle  and is cwere AR− . In linewere AR− ,48. In awere AR− . Howeverwere AR− . Moreovewere AR−  and aromwere AR− ,53 made In our study, the majority of Thai breast cancer patients had tumors that expressed the AR. These tumors were mostly hormonal and HER-2 positive in nature. Expression of the AR in triple negative breast tumors was less common. When compared with data from Western studies, the Thai patients show distinct differences in AR expression and the ratio between hormonal/HER-2 responsive and triple negative breast tumors. An inverse relationship between the non-hormone-dependent proliferation index Ki-67 and AR expression was found in this study. It is suggested that AR expression may be a prognostic factor for a more favorable outcome of the disease. Further experimental and human studies are needed to establish whether or not the AR is also a useful therapeutic target in a specific group of patients besides already available adjuvant systemic treatments with ER antagonists and aromatase inhibitors.


AR gene rearrangement promoting synthesis of constitutively-active truncated AR splice variants (AR-Vs)  = 69 nmol/L, 95% confidence interval [CI] = 53–89 nmol/L; AR1–651 [a constructed AR splice variant] IC50 = 309 nmol/L, 95% CI = 236–687 nmol/L in 22RV1 cells). Ailanthone decreased the androgen-dependent induction of endogenous AR downstream genes PSA, TMPRSS2, FKBP5, SLC45A3, and NDRG1 mRNA expression in LNCaP cells. To investigate whether ailanthone suppressed the functioning of AR-V which lacks the LBD, we performed RNA sequencing after treating LNCaP cells with or without ailanthone in the absence or presence of AR1–651. Indeed, ailanthone strongly suppressed AR1–651-induced expression of some genes, such as PSA and TMPRSS2, supporting the potential therapeutic use of ailanthone in CRPC. Moreover, ailanthone reduced the expression of both the full-length AR and the AR-Vs in vitro and in vivo. Using the sulforhodamine B colorimetric (SRB) assay, we confirmed that ailanthone potently inhibited the growth of several PCa cell lines including LNCaP, c4–2b, 22RV1, and LAPC4. Interestingly, ailanthone more potently inhibited the growth of AR-positive PCa cells than either AR-negative tumor cell lines or normal prostate cell lines. Notably, not only intraperitoneal injection administration but also per os administration of ailanthone had excellent efficiency for blocking the growth and metastasis of CRPC in LNCaP, 22RV1, and VCaP subcutaneous xenografts and 22RV1-luc orthotopic xenografts.To identify compounds that block the transcriptional activities of both ligand-dependent full-length AR (AR-FL) and AR-Vs, we used the MMTV-luciferase (MMTV-luc) reporter system that contains AR-binding elements  to screetivities . In our ications , ailanthOur study also explored the mechanism of ailanthone-induced AR degradation . We founIn pharmacokinetic studies, ailanthone exhibited good solubility in water and good bioavailability (>20%). We addressed some key safety issues of ailanthone, such as cytochromes P450 (CYPs) inhibition and hepatotoxicity. The current study showed that ailanthone had no obvious inhibitory effects on the main CYPs in humans and rats. In addition, ailanthone did not influence the expression of CYP enzymes and had no significant hepatotoxicity after a 5-day administration in the present study. Therefore, ailanthone would have a low potential to cause possible toxicity and drug–drug interactions in which CYP enzymes are involved, suggesting a sufficient safety window for its putative use as a promising anticancer agent. Meanwhile, various physicochemical properties of ailanthone calculated on the ACD/I-Lab showed that the physiochemical parameters of the natural compound ailanthone met with “Lipinski’s Rule of Five” . Our resIn conclusion, we screened and characterized ailanthone, a novel compound with excellent drug-like characteristics that is able to overcome MDV3100-resistance in PCa cell lines. Ailanthone was efficacious in suppressing the growth and metastasis of CRPC via targeting p23. As a result, ailanthone can be considered a new potential drug candidate for PCa, and it is worthy of further research and investigation.


Drosophila melanogaster germline stem cells (GSCs) to survive exposure to low doses of ionizing radiation (IR) as a model of adult stem cell injury and identified a regeneration defect in aging GSCs: while aging GSCs survive exposure to IR, they fail to reenter the cell cycle and regenerate the germline in a timely manner. Mechanistically, we identify foxo and mTOR homologue, Tor as important regulators of GSC quiescence following exposure to ionizing radiation. foxo is required for entry in quiescence, while Tor is essential for cell cycle reentry. Importantly, we further show that the lack of regeneration in aging germ line stem cells after IR can be rescued by loss of foxo.Aging stem cells lose the capacity to properly respond to injury and regenerate their residing tissues. Here, we utilized the ability of  Stem cells are unspecialized cells that have the unique ability to replace dead cells and repair damaged tissues. To give rise to new cells, stem cells need to divide. This process, known as the cell cycle, includes several stages and is regulated by many different genes.foxo helps cells respond to stress and to regulate the cell cycle and cell death. Defects in this gene have been linked to age-related diseases, such as cancer and Alzheimer’s disease. Previous research has shown that foxo can also regulate Tor – a gene that helps cells to divide and grow.For example, in many organisms, a gene called As we age, stem cells become less efficient at regenerating tissues, especially after exposure to toxins and radiation. However, until now, it was not known how stem cells control their division after injury and during aging, and what role these two genes play in injured and aging stem cells.foxo paused the cell cycle of the damaged stem cells. After 24 hours, Tor was able to overcome the action of foxo, and the stem cells resumed dividing and regenerating the damaged tissue. However, in old stem cells, foxo and Tor were misregulated and the stem cells could not restart dividing or repairing tissue after injury. When the levels of foxo in old stem cells were experimentally reduced, their ability to regenerate the tissue was restored.Now, Artoni, Kreipke et al. used germline stem cells from fly ovaries to investigate how young and old stem cells respond to injury. In young flies, foxo and Tor are misregulated during aging and how these two genes interact with each another. In future, this could help develop new anti-aging therapies that can restore the body’s natural ability to repair itself following injury. Moreover, since cancer cells can become resistant to conventional cancer treatment by withdrawing from the cell cycle, developing new treatments that target foxo and Tor could help beat cancer and prevent its reoccurrence.These discoveries provide new insights into how stem cells respond to injury and suggest that stem cell aging may be a reversible process. A next step will be to investigate why  In tissues with continuous cellular turnover, homeostasis is maintained by resident populations of adult stem cells. These cells both self-renew to maintain a constant pool of pluripotent cells and differentiate into a variety of cell types to replace cells that are lost to either natural wear and tear or to acute injury and insult . As tissDrosophila germline stem cells (GSCs) to survive IR, even when their progeny undergo rapid apoptosis. GSCs are resistant to the apoptotic effects of ionizing radiation . The fly is a particularly interesting model organism with which to examine stem cell survival post IR because recent work has demonstrated that there are several cell populations that display differing levels of resistance to ionizing radiation. Previous work in the young fly has shown a remarkable ability of adiation : when fladiation . After aadiation . Today, Drosophila melanogaster are a versatile system with which to study age related changes in regenerative potential  in direct contact with their somatic niche (Spradling 1993). These GSCs undergo asymmetric rounds of self-renewing divisions to give rise to a new stem cell and to a transiently amplifying cell (cystoblast) that undergoes four incomplete divisions generating an interconnected 16 cell cyst, of which one cell will eventually become an oocyte. Intricate interactions between GSCs and somatic cells allow for GSC maintenance in the niche , a ubiquitin ligase which mediates the internalization and subsequent activation of the Delta and Serrate Notch ligands in the germline, resulted in a complete loss of GSCs, even before exposure to ionizing radiation (data not shown), confirming the essential role of Notch signaling in GSC maintenance  and dissected their ovaries 1, 2 and 7 days post IR. GSCs in the expanded niche enter and exit quiescence in a timely manner . We then assayed Tor mutant GSC division capacity by analyzing the morphology of spectrosomes and the number of daughters produced at 1 and 2 days post-IR and compared them to control and unirradiated GSCs. We found that when Tor is knocked down, there is an even larger decrease in the percent of GSCs with elongated spectrosomes one day post-IR than in control animals, suggesting a higher penetrance in cell cycle exit , a downstream effector of TORC1. We compared levels of Foxo and p-S6 staining in young, wild type files following exposure to ionizing radiation. We observed a dramatic increase in the level of Foxo in GSCs’ nuclei 1 day post-IR  and dissect the mechanism for this process. Following exposure to IR, most aging GSCs survive, but fail to reenter the cell cycle and regenerate the germline, a process that is activated in young flies post IR. This is not due to a defect in DNA damage repair, as DNA damage repair concludes in a timely manner, even though the aging GSCs fail to return to the cell cycle. We have now identified two key regulators for IR induced quiescence: foxo and Tor. These two genes have opposing roles in regulating GSC cell cycle, exit and reentry after IR, respectively. Furthermore, Tor inactivation by RNAi or Rapamycin treatment induces a premature GSC aging phenotype, impairing Tor-dependent regeneration post injury. Conversely, knocking down foxo in aging animals rescues the aging phenotype, allowing GSCs to regenerate the germline, as observed in young flies. Finally, we show that foxo and Tor have opposing patterns of expression in the germarium and depletion of foxo leads to increases in Tor activity. This suggests that foxo regulates post-IR quiescence and cell cycle reentry by regulating Tor activity. Importantly, we show that loss of foxo rescues the GSC age-related regeneration phenotype due to IR. Overall, this study shows that IR induced quiescence is regulated by foxo and the mTOR ortholog, Tor, and suggests that upregulation of foxo and misregulation of Tor signaling in aging adult stem cells may be responsible for the decline in regenerative capacity following injury or insult  since rapamycin preferentially targets TORC1. We cannot completely rule out a role for Tor complex 2 (TORC2) in GSCs’ quiescence since rapamycin treatment has been shown to affect TORC2 activity by keeping Tor associated with TORC1 (Mechanistic target of rapamycin (mTOR) signaling has been implicated in a number of different age-related functions, from extension of lifespan  to germlth TORC1 . FurtherTor activity are unable to reenter the cell cycle post-IR, which is unlikely to be a general consequence of Tor inhibition inhibiting GSC division. In a number of different experiments, we observed a more pronounced defect in GSC proliferation in the context of recovery from injury post-IR than at baseline. This indicates that, while Tor might play a role in regulating stem cell division and self-renewal under normal physiological conditions, it likely has an additional injury-specific role in helping to replenish adult tissues that have been damaged, either by natural wear and tear or due to disease or injury. Given Tor’s ability to regulate translation, nucleotide synthesis, autophagy, lipid synthesis, and proteasome assembly,  in Drosophila and insulin-like growth factor (IGF1) in mice, result in Foxo activation and significant lifespan extension , which controls the G1 to S transition, and several ribosomal proteins, which are directly involved in protein translation, are misregulated in aging  to localize to the lysosome , P[UAS-Dcr-2.D]1, w1118; P[GAL4-nos.NGT]40 (RRID:BDSC_25751), UASp-foxoRNAi (RRID:BDSC_32427 and RRID:BDSC_32993), UASp-TorRNAi (RRID:BDSC_35578), UASp-ThorRNAi (RRID:BDSC_36815), UASp-dmRNAi (RRID:BDSC_43962), UASp-dapRNAi (RRID:BDSC_36720), UASp-LokiRNAi (RRID:BDSC_64482). The following stocks were previously generated for and described in Delta/CyO, UASp-Delta/CyO; Dad-GFP/TM3, UASp-Delta/CyO; Ly/TM3. The following stocks were previously generated for and described in dap4The following stocks were obtained from the Bloomington Drosophila Stock Center at Indiana University: w[1118]  of the following genotypes AB_528070 1:30), mouse anti-Lamin C (RRID:AB_528339 1:30) rabbit anti-γH2AV (RRID:AB_828383 1:200), rabbit anti-p-S6 (RRID:AB_916156 1:200), rabbit anti-foxo (generous gift from Pierre Léopold 1:200). Ovaries were incubated with primary antibodies for either 1.5 hr at room temperature or overnight at 4°. After washes with PBT, secondary fluorescence antibodies were utilized including anti-rabbit Alexa 488 (RRID:AB_221544 1:250) and anti-mouse 568 (RRID:AB_2535773 1:250) for 1.5–2 hr at room temperatures in the dark. DAPI was added to one of the final washes to visualize cells’ nuclei. The samples were mounted in glycerol and analyzed on a Leica SPE5 confocal laser-scanning microscope.Ovaries were fixed in 4% paraformaldehyde for 15 min, rinsed in PBT (PBS containing 0.2% Triton X-100), and blocked in PBTB  for at least one hour at room temperature. Samples were stored up to 72 hr at 4° in PBTB. The following primary antibodies were used: mouse anti-adducin (RRID:All data are presented as the mean of at least three independent experiments (n ≥ 3) with the standard error of the mean (SEM) indicated by error bars, unless otherwise indicated. Statistical significance was determined using Student's t test (for two groups) or ANOVA with the appropriate post hoc test (for more than two groups). Data were compiled using Excel 2013 software and analyzed using Excel  or the Astatsa Online Web Statistical Calculator . In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.Drosophila germline stem cells" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Fiona Watt as the Senior Editor and Reviewing Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Loss of FOXO rescues stem cell aging in The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Kreipke et al. examine the mechanism by which exposure of germline stem cells (GSC) to ionizing radiation (IR) leads to a transient block in cell cycle progression followed by subsequent regeneration of the germline in young fruit flies. In contrast IR triggers a permanent inhibition of germline production in older flies but does not eliminate the GSC, suggesting that there is some form of regulation of exit and re-entry of the cell cycle for young but not older germlines. Using a combination of RNAi knock-downs and histochemical staining the authors analyze the mechanistic basis for the regulation of the GSC entry/exit from the cell cycle and find that the stress induced transcription factor Foxo and the metabolic homeostasis/aging pathway regulator Tor play key opposing roles in this process. Following IR exposure Foxo levels increase and inhibit Tor signaling. Tor signaling is required for post-IR treated quiescent GSCs re-entering the cell cycle, while Foxo induces exit into the quiescent state. Perhaps the most interesting result, which has potential practical implications for combatting Tor-dependent aging, is that RNAi knock-down of Foxo can rescue quiescent GSC in older flies resulting in regeneration of a germline. These conclusions are well supported by high quality data and should be of broad interest to readers in the fields of stem cell biology and aging.Essential revisions:Below are listed minor revisions that the reviewers request in order to improve the clarity of the paper.1) In the second paragraph of the subsection “Aging germline stem cells survive exposure to ionizing radiation, but fail to re-enter the cell cycle in a timely fashion”, the authors indicate that the majority of germaria still had GSCs 7 days after irradiation. This is correct but at both 4 and 6 weeks a decrease in numbers of GSCs is evident. Can the authors please comment?2) In 3) The loss of GSCs after irradiation in germaria with ectopic Delta  should b4) It would be informative if Tie activation could be shown only in cells that border daughters in 5) The way data are displayed in 6) The increase in Foxo staining in 7) The increase in p-S6 staining in 8) In 9) In Further points:1) 2) Foxo staining should always be referred as "Foxo" not "foxo". Essential revisions:1) In the second paragraph of the subsection “Aging germline stem cells survive exposure to ionizing radiation, but fail to re-enter the cell cycle in a timely fashion”, the authors indicate that the majority of germaria still had GSCs 7 days after irradiation. This is correct but at both 4 and 6 weeks a decrease in numbers of GSCs is evident. Can the authors please comment?Drosophila females will lose GSCs during late stages of aging process. The small loss of GSCs observed at 4 and 6 weeks is an early manifestation of this process.As previously described, 2) In The decrease in germaria with >4 progeny reflects the small loss in GSC number observed in aging animals.3) The loss of GSCs after irradiation in germaria with ectopic Delta  should bWe have now quantified GSCs loss 1 day following IR in germaria with ectopic Δ and added the information in 4) It would be informative if Tie activation could be shown only in cells that border daughters in Drosophila P-Tie antibody exists to our knowledge.While this is an important question, at this time Tie activation is difficult to show directly since no reliable 5) The way data are displayed in To provide the opportunity for the reader to determine the decrease in WT GSC with elongated spectrosomes we have now included the data used for the graph in 6) The increase in Foxo staining in We show a high magnification image of 6-week old GSCs stained for Foxo see . However7) The increase in p-S6 staining in We have now quantified the P-S6 increase in FOXO KD vs. control in aged animals. The increase of P-S6 in FOXO KD animals is significant, suggesting that FOXO based aging phenotype manifests through Tor and its target P-S6.8) In We have now analyzed foxo>RNAi without GAL4. No obvious defects were observed see .9) In We quantified the offspring of 6-week old Foxo RNAi flies before and after IR. We did not observe a rescue of the offspring phenotype in wild type 6-week old flies. As suggested by the reviewer, other effectors might also be essential for full germline regeneration.Further points:1) We have now corrected this statement in the subsection “foxo represses Tor in GSC after IR”.2) Foxo staining should always be referred as "Foxo" not "foxo".We have now corrected this.


Non-alcoholic fatty liver disease (NAFLD) given its association with obesity and diabetes may perhaps exert distinct free fatty acids (FFA) pattern, but the understanding of this phenomenon is limited. To this effect, we evaluated FFA profiles among healthy subjects and NAFLD patients stratified by body weight, to identify FFA valuable for early diagnosis of NAFLD.Serum FFA profiles of healthy and NAFLD  subjects was determined using gas chromatography–mass spectrometry (GC–MS) and distinctions in FFA patterns were evaluated using one-way ANOVA while Receiver operating characteristics (ROC) and logistic regression models were used to explore FFA significant for diagnosing NAFLD.P < 0.05) serum FFA profiles compared to healthy controls (HC). While total FFA profiles were insignificantly different between lean (2093.33 ± 558.11 μg/ml) and overweight (2420.81 ± 555.18 μg/ml) NAFLD patients, obese NAFLD (2739.01 ± 810.35 μg/ml) presented most significantly elevated (P < 0.05) total FFA profiles compared with HC. Of the four FFA; myristic acid (14:0), palmitoleic acid (16:1), γ-linolenic acid (γ-18:3) and cis-7,10,13,16,19-docosapentaenoic acid (22:5), selected in ROC analysis given their high Youden’s index and AUC, only 14:0; 5.58 and 16:1; 4.36 had statistical significant odd ratios.NAFLD patients presented significantly higher (Our findings suggest 14:0 and 16:1 are promising for early diagnosis of NAFLD.The online version of this article (doi:10.1186/s12944-017-0551-1) contains supplementary material, which is available to authorized users. Serum free fatty acids (FFA) perhaps reflect the imbalance between release and uptake of triglyceride (TG) in muscle, heart, liver, and other tissues , and itsn-3 polyunsaturated fatty acids (LCPUFA) are beneficial against fat deposition and liver steatosis. Depletion of LCPUFA (n-6 and n-3 series) has been linked with lipid accumulation [Elevated plasma FFA in NAFLD patients has been mostly clarified to be closely associated with metabolic changes in the liver –8. For emulation , 12. Barmulation  evaluatemulation , it is yEven though obesity is a well-known risk factor for the onset of NAFLD, yet NAFLD could also be observed in normal weight individuals  with unique metabolic characteristics such as lower fasting blood glucose, HbA1c, blood pressure and better insulin sensitivity when compared with obese patients. It has also been considered as a frequent cause of cryptogenic liver disease in normal weight subjects . To dateAgainst this background, we applied metabonomics to measure the FFA profile of NAFLD patients primarily to evaluate distinctions in FFA profiles of NAFLD patients by body weight as well as promising FFA for timely diagnosis of NAFLD.Of the 80 healthy individuals and 240 NAFLD patients recruited at Physical Examinations Center of the Second Affiliated Hospital, Harbin Medical University between March 12, 2013 and April 20, 2013, individuals with a history of alcohol drinking, malignancies, pregnancy, and long-term use of estrogens, tamoxifen or corticosteroids were excluded from the current study [Additional file 2, with normal liver biopsy and without a clinical diagnosis of any known liver disease or another disease(s).Healthy controls (HC): Body mass index (BMI); 18.5–24.0 kg/m2.Lean NAFLD: Fulfils the diagnostic criteria for NAFLD only, without any history or diagnosis of any other disease(s) and BMI: 18.5–24.0 kg/m2.Overweight NAFLD: Fulfils the diagnostic criteria for NAFLD only, without any history or diagnosis of any other disease(s) and BMI: 24.0–28.0 kg/m2.Obese NAFLD: Fulfils the diagnostic criteria for NAFLD only, without any history or diagnosis of any other disease(s) and BMI ≥ 28.0 kg/mIn all, 66 HC and 170 NAFLD patients; lean – NAFLD (67), overweight – NAFLD (55) and obese – NAFLD (48) met the inclusion criteria and were included in final analysis. Also, the Ethics committee of Harbin Medical University approved the study (2013019) and written informed consents were obtained from all respondents prior to their voluntary participation in the study.Subjects who participated in the physical examination were interviewed by trained interviewers to complete a questionnaire that included questions such as; age, sex, history of disease(s), drug use, regular exercise, and alcohol consumption. Regular exercise was categorised as ‘vigorous’ or ‘moderate’ or ‘none’ where respondent reportedly engaged in any form of strong/hard physical exercise/labour  at leisure or occupational time for ≥3 times or once or none per week respectively. Habitual alcohol intake was assessed using the amount of alcohol intake of respondents multiplied by the frequency of consumption.Weight, height, waist circumference (WC), body fat and blood pressure (BP); including systolic BP (SBP) and diastolic BP (DBP) were measured by well-trained examiners as reported in . Body maSerum FFA profile was measured using gas chromatography–mass spectrometry method (GC-MS) described previously in .Firstly, we purchased 12 fatty acid standards from Sigma , including myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1n-7), stearic acid (18:0), oleic acid (18:1n-9), linoleic acid (18:2n-6), linolenic acid (18:3n-3), γ-linolenic acid (γ-18:3n-6), arachidonic acid (20:4n-6), cis-5,8,11,14,17-eicosapentaenoic acid (20:5n-3), cis-7,10,13,16,19-docosapentaenoic acid (22:5n-6), and cis-4,7,10,13,16,19-Docos-ahexaenoic acid (22:6n-3). Stock solutions of all the above fatty acids, internal standards (heptadecanoic acid) and calibration samples (spiking with 12 different concentrations of fatty acids standards) were prepared.2SO4 was added to deposit protein. FFA were extracted using 3 mL ethyl acetate, mixed for 60s (using a vortex mixer) and centrifuged at 4000×g for 10 min at room temperature. The ethyl acetate phase was evaporated to dryness under N2. Following the addition of 2 mL 10% H2SO4-CH3OH and incubation in 62 °C water bath for 2 h, 2 mL saturated sodium chloride and 2 mL hexane was sequentially added and mixed for 60s to obtain the fatty acid methyl esters. Samples were evaporated to dryness under N2 gas, and 100 μL hexane was added to each tube prior to analysis.Briefly, aliquots (200 μL) of serum were spiked with an internal standard working solution (200 μL heptadecanoic acid C17:0200 μg/mL), and 1 mL 0.05% HFFA concentrations of all standard and serum samples were determined using TRACE gas chromatograph with a Polaris Q mass spectrometer  (GC–MS). A split injector (the split ratio being 1:10) at 230 °C was used to add the sample (1.0 μL) onto a J&W DB-WAX  capillary column and fatty acid methyl esters were separated at constant flow . The ionNAFLD in this study was defined using the guidelines provided by the Chinese Association for Study of Liver Diseases . BrieflyIn this study, NAFLD is diagnosed provided histopathological reports of liver biopsies fulfills the pathological diagnostic criteria for fatty liver disease in the absence of; (a) specific disease(s) that could trigger liver steatosis; viral hepatitis (HBV/HCV), hepatolenticular degeneration, autoimmune diseases or/and a history of total parenteral nutrition, or intake of hepatotoxic-inducing drugs , (b) history of habitual alcohol consumption or ethanol intake of <140 g/week or <70 g/week for men and women respectively.2), overweight – NAFLD (BMI: 24.0–28.0 kg/m2) and obese – NAFLD (BMI ≥ 28.0 kg/m2).Furthermore, patients who satisfy the above criteria for NAFLD in our study were additional categorized by BMI i.e. lean – NAFLD (BMI: 18.5–24.0 kg/mAll data were presented as mean ± standard deviation (SD) and proportion. Continuous and categorical variables were analysed by one-way ANOVA and Chi-square test respectively. Distinction in FFA patterns among HC, lean –, overweight – and obese – NAFLD patients were explored using one-way analysis of covariance (ANCOVA) adjusting for age and sex in addition to Bonferroni post hoc test to investigate distinctions between any two groups. Partial correlation analysis was adopted to explore the relationship between FFA and biochemical indicators, after adjusting for age and sex, in all participants. Receiver operating characteristics (ROC) analysis was used and areas under the ROC curve (AUC) were calculated to compare the effectiveness of different FFA to identify NAFLD. Logistic regression models were adopted to evaluate the odd ratio (OR) and 95% confidential interval (CI) of FFA with higher AUC and Youden’s index (the first three) for NAFLD of higher concentrations than their cut-off values.P value <0.05 (two-tailed) was considered statistically significant.SPSS 18.0  was used for statistical analysis and a Table Additionally, biochemical indicators were insignificantly different between lean and overweight NAFLD subjects, but fasting insulin and HOMA-IR were significantly higher and HDL-c was significantly lower among obese-NAFLD than lean-NAFLD or overweight-NAFLD.The GC-MS method had excellent linearity and repeatability and bias% are within acceptable range. Serum profiles of FFA in HC and NAFLD patients are summarised in Table Twelve FFA alongside total FFA positively correlated with FBG, blood lipid  and liver enzymes   are presented in Table The OR 95% CI) for NAFLD of 4 FFA; 14:0, 16:1, γ-18:3 and 22:5 are summarized in Table % CI for In the current study, we identified four groups according to subjects’ BMI and NAFLD status , and firstly analysed the FFA profiles of all NAFLD patients and HC. All NAFLD patients had higher serum concentrations of all types of and total FFA than HC. Lean and overweight NAFLD had similar concentrations in all FFA, but obese NAFLD patients presented significantly higher levels of all types of FFA. All FFA were positively associated with glucose, ALT, GGT, TC and TG, (independent of age and sex). These biochemical indices are likely important in the diagnosis of NAFLD. In addition, we observed 14:0 and 16:1 as unique FFA of prominent diagnostic importance for early screening NAFLD.NAFLD is the most common spectrum of liver disease in many countries . It is aIn the current study, serum FFA profiles were significantly higher in NAFLD patients and positively associated with almost all metabolic indicators, especially blood glucose, lipid and liver enzymes. The positive association between serum FFA, fasting insulin and HOMA-IR may be explained by the inhibitory effects of FFA on glycogenolysis, thus leading to elevated insulin levels and hepatic IR .On the other hand, persistent elevation of FFA is likely to also increase gluconeogenesis  and inhislc7a3a) – null mutants zebra fish hepatocyte and fasted cultured human hepatocytes. slc7a3a is one of the many kinds of specialized protein on the plasma membrane vital for intracellular transport with inhibited expression during the fasting. As a result, nitric oxide – mediated cyclic guanosine mono phosphate synthesis which acts upstream of adenosine mono phosphate (AMP) – activated protein kinase to regulate lipid oxidation are inhibited thereby leading to anomalous lipid retention in the hepatocytes [Comprised hepatic lipid uptake has been linked to severe lipid accumulation in hepatic cells. In a recent report by Gu et al. , hepaticatocytes .Among the 12 FFA evaluated in our study, 14:0 and 16:1 proves more effective than other FFA in predicting NAFLD. 14:0 is a long chain saturated fatty acid, predominantly produced from fatty acid synthase (FAS) pathway. A small proportion of 14:0 is synthesised from the shortening of 16:0 by peroxisomal β-oxidation . It coul16:1 is a product of stearoyl-CoA desaturase (SCD), and could reflect the activity of SCD. Circulating 16:1 is associated with endogenous sources  of fatty acid synthesis . It is sDietary pattern exposure is a vital component as to either improving or compromising vascular health. In a review by Scicchitano et al. , the benIn tandem with our finding, diary fat – a rich source of 14:0 has been reported to acutely raise LDL levels. Zock et al.  in a 3-wThere are some limitations worth mentioning in our current study. Both 14:0 and 16:1 can be derived from diets and whether their dietary intakes were associated with serum levels were not studied. Even though FFA profiles stratified by sex were insignificantly different  and 16:1  are of promising diagnostic value in the early diagnosis of NAFLD especially among normal weight individuals.


The objective of this study was to detect differences in the distribution of the left and right ventricle (LV & RV) activation rate (AR) during short-duration ventricular fibrillation  and long-duration ventricular fibrillation VF  in normal and heart failure (HF) canine hearts.Ventricular fibrillation (VF) was electrically induced in six healthy dogs (control group) and six dogs with right ventricular pacing-induced congestive HF (HF group). Two 64-electrode basket catheters deployed in the LV and RV were used for global endocardium electrical mapping. The AR of VF was estimated by fast Fourier transform analysis from each electrode.In the control group, the LV was activated faster than the RV in the first 20 s, after which there was no detectable difference in the AR between them. When analyzing the distribution of the AR within the bi-ventricles at 3 min of LDVF, the posterior LV was activated fastest, while the anterior was slowest. In the HF group, a detectable AR gradient existed between the two ventricles within 3 min of VF, with the LV activating more quickly than the RV. When analyzing the distribution of the AR within the bi-ventricles at 3 min of LDVF, the septum of the LV was activated fastest, while the anterior was activated slowest.A global bi-ventricular endocardial AR gradient existed within the first 20 s of VF but disappeared in the LDVF in healthy hearts. However, the AR gradient was always observed in both SDVF and LDVF in HF hearts. The findings of this study suggest that LDVF in HF hearts can be maintained differently from normal hearts, which accordingly should lead to the development of different management strategies for LDVF resuscitation. Frequency analysis using the fast Fourier transform (FFT) has been widely used to characterize features of ventricular fibrillation (VF). Electrical and optical mapping experiments in previous animal and human studies have evaluated the spatiotemporal distribution of the activation rate (AR) during VF and have provided mechanistic insight into the organization of VF, thereby improving our understanding of the initiation and maintenance of this complex arrhythmia –7. The rThe activation of VF changes as it continues, which raises the possibility that the relative importance of different arrhythmogenic mechanisms changes. It has been proposed that defibrillation mechanisms and efficacy may differ in different pathological animal models during different stages of VF . PanfiloTwelve beagles (11 ± 1.2 kg) were divided into two groups. Six dogs were selected to create the HF model, and the other six dogs served as the control group. A pacemaker  was implanted in a subcutaneous pocket and attached to a pacing lead  in the RV apex under fluoroscopic visualization via the right external jugular vein. When the surgery was completed, the dogs were given antibiotics, after which they underwent rapid ventricular pacing at 240 beats per minute for three to 4 weeks. Then, echocardiography was performed to confirm that the HF model was successfully established. Rapid pacing was maintained until a day before the electrophysiological mapping study.Each animal was injected intramuscularly with ketamine 10 mg/kg) and tropine (0.04 mg/kg) for anesthetic induction. Anesthesia was maintained intravenously with propofol (8–16 mg/kg/h), and the animals were ventilated in a restrained, dorsally recumbent position. To determine the adequacy of the anesthesia, ventilation and oxygenation, the arterial blood pressure, blood gases, cardiac electrical activity, body temperature and serum electrolytes were monitored, and interdigital reflexes were tested throughout the entire study. After the completion of the data collection, the animals were euthanized with an intravenous injection of potassium chloride. The hearts were exposed through a median sternotomy and supported in a pericardial sling. A catheter  was inserted for defibrillation with the negative electrode in the RV apex and the positive electrode in the superior vena cava  was introduced through the left carotid artery into the LV. Another basket catheter was inserted through the right jugular vein into the RV. Each catheter contained eight splines each with eight (8 × 8) electrodes approximately 2 mm apart. These two catheters were used to map the ventricular endocardium simultaneously  through one of the basket electrodes. Four VF episodes were induced in each animal. The first three VF episodes were recorded for 20 s as a short-duration (SDVF) episode before being halted by a 400–600 V biphasic shock (6/4 ms) delivered from a defibrillator electrode. The last VF episode was allowed to continue for at least 3 min , and the animal was not resuscitated. The first 20 s of VF data during this LDVF episode served as the fourth SDVF episode. There were no significant differences in the VF parameters among multiple short VF episodes. Additionally, the AR distribution did not depend on the basket electrodes used to induce VF. The two 64-electrodes of the basket catheter and the six limb-ECG leads were recorded with a 160-channel cardiac data acquisition system. The AR was estimated through FFT analysis of VF at each electrode of the basket catheter and the six limb leads of the body ECG for a 2 s interval beginning 20 s after VF induction. The frequency with the highest power between 1 and 20 Hz was taken as the AR.t test. A value of P<0.05 was considered significant.To ascertain how the AR in different ventricular regions varied during VF, the endocardium of the two ventricles were divided into 8 zones according the display of the basket electrode Fig. . We defiP < 0.0001), accompanied by significant increases in the LV end-diastolic dimension and LV end-systolic dimension.There was unambiguous evidence  of myocardial systolic dysfunction in the HF group but not in the control animals. The LV ejection fraction for the HF canines was substantially decreased  in the first 20 s. However, there was no significant difference in the AR between LV and RV after 20 s. There was a dramatic decrease in AR between 20 s and 90 s, after which it declined smoothly. At 3 min, there was no significant difference between the LV and RV  . When analyzing the distribution of AR within the RV, the posterior wall activated the fastest  ; and RV apex>RV base   Fig. , while t27) Fig. , with a 01) Fig. .Fig. 2EvP = 0.02). At 3 min, the LV still activated more quickly than the RV  . When analyzing the distribution of the AR within the RV, the septum of the RV was found to be activated the fastest  ; and RV apex>RV base   Fig. , while t04) Fig. , with a 01) Fig. .P = 0.015). Additionally, the LV-RV AR gradient was greater at the beginning than at the end of the entire VF episode in the HF group   In the control group, the LV activated slightly faster than the RV in the first 20 s, after which there was no difference in the AR between the LV and RV. At 3 min of LDVF, the posterior of the LV activated fastest, while the anterior activated the slowest. The apical part activated faster than the basal part in the bi-ventricles. (2) In the HF group, there was always a noticeable LV-RV AR gradient, with the LV activating more quickly than the RV. At 3 min of LDVF, the septum of the LV activated fastest, while the anterior activated the slowest. Similarly, the apical part activated faster than the basal parts in the bi-ventricles. (3) In both groups, the AR decreased dramatically for the first 90 s, after which it declined more smoothly. (4) The AR of both the RV and LV in the HF group were slower than those in normal hearts for the first 90 s. From 90 s to the end of the analysis at 3 min of VF, neither the RV nor LV in HF hearts was activated differently from the bi-ventricles of the control animals.Data from previous animal studies suggest that one or two primary wavefronts located in the regions with the fastest AR drive the rest of the heart and that these regions, rather than the entire myocardium, are responsible for the maintenance of VF .In the control group of this study, the overall wavefront direction of the bi-ventricles was from the apex to the base and from the posterior to the anterior at 3 min of LDVF. Previous studies have shown that the posterior LV activates faster than the anterior LV during VF in swine and that VF wavefronts tend to move from the posterior to the anterior. These reports are consistent with our findings , 13. TheIn the HF group at the 3 min LDVF, we demonstrated that the AR of RV was much slower than the LV and that the septum of the LV activated more rapidly than any other portion of the LV or the RV, indicating that if there existed a mother rotor, it was probably located in the septum. This hypothesis is supported by the findings of Ikeda et al., who studied endocardial activation patterns during VF, in which the septum was found to have more wavelet numbers and a shorter cycle length than the other portions of the LV and RV . It has k1), with the left ventricular myocytes showing significantly weaker inward-directed rectification than the right ventricular myocytes, has been postulated to play crucial roles in the LV-RV gradients of excitation frequency during VF [Rogers et al.  describeuring VF , 21. In In the HF dogs, there was always an AR gradient between the bi-ventricles as VF progressed. The gradient indicated that these regions participated in different ways in different animal models. For previous studies with diseased hearts, Huang et al.  demonstrThe findings from our study demonstrated that there were regional differences in the different portions of the LV and RV, with some regions activating rapidly and other regions activating slowly. We observed a significant tendency for the VF wavefronts to spread from the apex toward the base of both the LV and RV in the normal and HF hearts. However, Nanthakumar et al.  did not A previous study reported that the incidence of out-of-hospital VF has declined compared with asystole as the initially recorded rhythm . CardiacIn the clinical setting, patients with preexisting HF are typically assumed to have a lower chance of successful cardiopulmonary resuscitation. The findings of this study indicate that LDVF is maintained differently in HF hearts than it is in normal hearts. Various intra- and inter-ventricular areas were activated differently during LDVF under the condition of HF. Some areas might be triggered by early or delayed after-depolarization with increased AR. Thus, in addition to electrical shock, other treatments, including agents that inhibit triggered activities and pacing, could potentially improve the outcomes of cardiopulmonary resuscitation in such patients.The limitations of this study are as follows. First, although the global bi-ventricular endocardium was simultaneously mapped, our mapping technology could not detect the micro-reentry at the endocardium. The basket electrodes were widely spaced, such that the fine details of the endocardial activation sequence were not identified. Second, transmural mappings were not performed. Therefore, this study could not detect the presence of a transmural AR gradient between the epicardium and endocardium during VF.Estimates of the distribution of VF AR are not uniform across the endocardium. A global bi-ventricular endocardial AR gradient was observed in the first 20 s of SDVF but was not observed in LDVF in normal hearts. However, the AR gradient was always present in both SDVF and LDVF in the HF hearts. The findings of this study suggest that the LDVF in the HF hearts is maintained differently from normal hearts, which accordingly leads to different recommended management strategies during LDVF resuscitation.


Rehabilitation of marine turtles in Queensland has multifaceted objectives. It treats individual animals, serves to educate the public, and contributes to conservation. We examined the outcome from rehabilitation, time in rehabilitation, and subsequent recapture and restranding rates of stranded marine turtles between 1996 and 2013 to determine if the benefits associated with this practice are cost-effective as a conservation tool. Of 13,854 marine turtles reported as stranded during this 18-year period, 5,022 of these turtles were stranded alive with the remainder verified as dead or of unknown condition. A total of 2,970 (59%) of these live strandings were transported to a rehabilitation facility. Overall, 1,173/2,970 (39%) turtles were released over 18 years, 101 of which were recaptured: 77 reported as restrandings  and 24 recaptured during normal marine turtle population monitoring or fishing activities. Of the turtles admitted to rehabilitation exhibiting signs of disease, 88% of them died, either unassisted or by euthanasia and 66% of turtles admitted for unknown causes of stranding died either unassisted or by euthanasia. All turtles recorded as having a buoyancy disorder with no other presenting problem or disorder recorded, were released alive. In Queensland, rehabilitation costs approximately $1,000 per animal per year admitted to a center, $2,583 per animal per year released, and $123,750 per animal per year for marine turtles which are presumably successfully returned to the functional population. This practice may not be economically viable in its present configuration, but may be more cost effective as a mobile response unit. Further there is certainly benefit giving individual turtles a chance at survival and educating the public in the perils facing marine turtles. As well, rehabilitation can provide insight into the diseases and environmental stressors causing stranding, arming researchers with information to mitigate negative impacts. Caretta caretta) genetic stock and the southern and northern Great Barrier Reef green turtle (Chelonia mydas) populations . For example, the “Code of Practice—Care of Sick, Injured or Orphaned Animals in Queensland”  is availAs general public awareness for wildlife conservation has increased, there has been a corresponding increase in the numbers of stranded turtles reported, rehabilitated and subsequently released back into the wild with the intent of enhancing wild populations . AlthougTherefore, given the resources used in rehabilitating marine turtles, assessing the capacity of these species to readapt to the wild, including their ability to survive and reproduce, are essential to guarantee that resources allocated are maximising the number of marine turtles contributing to the functional population . QueenslWe investigated whether different causes of stranding as well as the length of time an animal spends in care influenced the long term survival of individuals during and post rehabilitation. We summarised and analysed the available data to provide rehabilitation facilities with options to undertake this method of species conservation.https://www.ehp.qld.gov.au/wildlife/caring-for-wildlife/strandnet-reports.html).Data used in this study were obtained through StrandNet, the Queensland Government’s Department of Environment and Heritage Protection (EHP) statewide database reporting threatened stranded marine turtles for the entire coast of Queensland and adjacent Commonwealth waters as outlined in Additional data were obtained from the EHP Queensland Turtle Conservation Project (QTCP), SeaWorld, Australia Zoo Wildlife Hospital and Underwater World-SEA LIFE Aquarium. The data provided by SeaWorld, Australia Zoo Wildlife Hospital and Underwater World-SEA LIFE Aquarium were used to complete data in StrandNet such as outcomes, causes of death and duration of care.The QTCP database is the Queensland Government’s EHP statewide database which records tagging and tag recaptures for all marine turtles encountered for the entire coast of Queensland and adjacent Commonwealth waters. Records are received from members of the public, trained volunteers and employees of EHP, QPWS, DAF and GBRMPA. Additional tag recoveries are reported by members of the public. Amalgamating these databases produced the first comprehensive dataset of strandings, causes and captures throughout Queensland for 1996 to 2013.Biometrics were assessed using standard measurements, gonad examination and/or dichotomous key characteristics. Age classes were broken down into three broad categories: small immature, large immature and adult-sized based on curved carapace measurements adapted from Cause of stranding was identified by examining information compiled from first-responders and trained staff who reviewed reports, photos and codes recorded in StrandNet . All detstranding is used to incorporate all reported sick, injured, incapacitated or dead marine turtles that either were found washed ashore or encountered at sea. It includes turtles which were entangled in synthetic debris including fishing nets and line, as well as turtles which were rescued .Disease is classified as turtles which exhibit protracted ill health from a cause consistent with a physiological condition and not otherwise caused by anthropogenic activity . This is often linked with poor body condition.Buoyancy disorder is used to describe turtles which were observed floating and in which no other presenting problem or disorders were observed.Unknown cause of death/stranding was used when a cause could not be accurately determined. In most cases for cause of death this was due to there being no necropsy performed and no obvious external cause identifiable with a gross examination.survivorship is defined as a turtle being found in good condition at least once after release from rehabilitation. Determination of condition was made based on coming onto a nesting beach or laparoscopic examination of the gonads or via in-water population surveys and being found to be in good condition at each capture. Rehabilitation was deemed non-successful if the animal was reported stranded again  within the timeframe of the collected dataset (1996–2013).For this study, Duration in care was calculated by subtracting the date of outcome, from the original date of admission/stranding.Rodeo is a technique developed to capture turtles during in-water surveys. This technique is presented pictorially in Turtles were included if they stranded along the east Queensland coast within the area of latitude −10.78° (Cape York) to −28.16°  and longitude 142.15 to 155° . RecaptuAnimals were matched between the databases using unique identifiers, such as titanium tag numbers. To find subsequent recaptures of the same animal, queries were performed with the capture date that was greater than the first recorded stranding.Outcomes were analyzed two ways: using the actual calculated time until outcome and then grouping time in care into three groups: admission to 7 days in care (short term stay), 7 to 28 days in care (medium term stay) and greater than 28 days in care (long term stay).R was used to perform all statistical analysis described above . ResultsWhen analyzing time period between release and recapture numbers are expressed as averages, ± a standard deviation.The three main rehabilitation facilities in Queensland were approached and asked to provide their estimated annual costs for marine turtle rehabilitation. These costs are derived based on pool maintenance, food, labor and some medical costs. They do not identify general operating costs that are absorbed by the facility. At best, they should be viewed as an estimate.The three facilities rehabilitated approximately 68% of turtles admitted to rehabilitation in Queensland. The costs supplied were then extrapolated out to account for all rehabilitated turtles in Queensland, at best these costs should be viewed as a low estimate. Calculations were then made based on numbers of turtles admitted to rehabilitation, numbers of animals released from rehabilitation, numbers of turtles encountered again, and numbers of unsuccessful attempts at rehabilitation.R2 = 0.70) and the proportion (R2 = 0.80) of stranded turtles which were sent to rehabilitation facilities in Queensland  were admitted to a rehabilitation facility. There was an increase over time in the number  were treated at three institutions: SeaWorld received 596 , Australia Zoo Wildlife Hospital received 788 , and Underwater World received 637 .Two thousand and twenty-one  and loggerhead turtles  with the other species , olive ridley turtles, Lepidochelys olivacea , Unknown , black turtle, Chelonia mydas agassizi ) remaining at low levels.Green turtles were most often sent to rehabilitation both by number and by proportion . This was followed by hawksbill turtles, Consistently, over the years, the majority of turtles sent to rehabilitation were small immature sized turtles . The numbers of large immature  and adult sized  turtles admitted varied each year.The most common cause of stranding for animals sent to rehabilitation was unknown . The most common identified presenting problems or disorders were disease , buoyancy disorder  and fracture . Irrespective of age class, the most common record of stranding for turtles sent to rehabilitation by proportion was from unknown reasons, followed by disease and buoyancy disorder .For all species with the exception of the black and flatback turtles, the most common cause of turtles being sent to rehabilitation was for unknown reasons followed by disease then buoyancy disorders . For logR2 = 0.47), while the proportion of turtles which died while in care decreased (R2 = 0.38). Between 1996 and 2013, the proportion of turtles which were released was highly variable with a slight decrease (R2 = 0.07).Between 1996 and 2013, there were changes in the outcomes of turtles sent to rehabilitation . OverallBetween 2005 and 2009, there was a steady increase in the proportion of turtles euthanized which reversed after this time. During this same period there was an inversely proportional decrease in the number of unassisted deaths in care . Of the R2 = 0.07), while the proportion of animals which were released decreased over time (R2 = 0.07). Both patterns showed a lot of variability.When combining the proportion of animals which died while in care (assisted and unassisted) and comparing against the number of animals which were released, the proportion of animals which died slightly increased over time . Overall, 500 rehabilitated turtles were euthanized. The cause of stranding for the highest number euthanized were unknown , followed by disease  and fractures .Between 1996 and 2013, 1,297 stranded and rehabilitated turtles died unassisted while in care. The highest number of these turtles died for unknown reasons , followed by disease  and fractures .Between 1996 and 2013, 1,173 stranded and rehabilitated turtles were released from rehabilitation alive. The number of turtles which were released for each stranding cause was variable, including unknown reasons , buoyancy disorder , and disease .Two thousand four hundred and ninety four stranded and rehabilitated turtles had duration of care recorded , 1,139 of which died unassisted in care (45.7%), 480 were euthanized (19.2%) and 875 were released (35.1%). When analyzing duration of care across all outcomes this was compared as a combined total of turtles sent to rehabilitation.Between 1996 and 2013, 25.4%  of all turtles died without assistance within the first 7 days, 11.3%  died between days 7 and 28, 8.9%  died after the first 28 days . In 1997Between 1996 and 2013, of the 1,173 turtles released from rehabilitation, 101 turtles were recaptured . This reOf the turtles released from rehabilitation and subsequently recaptured, 76.2%  were recorded as restranded and 17.8%  of them were recorded during normal population studies. Of the remaining six turtles, 5.9%  were recaptured during fishing activities.Twenty of the turtles that subsequently restranded were dead , 11 were alive and subsequently euthanized , 13 were alive and subsequently died unassisted  and 33 were alive and re-admitted for rehabilitation .The most common original cause of stranding for turtles that were recaptured alive was buoyancy disorder ; followed by unknown original cause of stranding ; all other original causes contributed a total of 31%. For the nine identified categories of restranding; seven of these categories , and unknown causes) showed there was more than a 50% chance the turtle would restrand for the same reason as it originally stranded .Collectively, SeaWorld, Australia Zoo Wildlife Hospital and Underwater World-SEA LIFE Aquarium reported spending a total average of $AUD112,000 per annum on marine turtle rehabilitation to treat approximately two thirds (68%) of all marine turtles received for rehabilitation. Extrapolating from the annual amount spent at SeaWorld, Australia Zoo Wildlife Hospital and Underwater World-SEA LIFE Aquarium and assuming that other facilities have a similar expenditure, this approximates $AUD165,000 being spent per annum in Queensland across the participating rehabilitation facilities on marine turtle rehabilitation.Over the 18 years of this study, 2,970 turtles were admitted to all rehabilitation facilities in Queensland, equating to an average of 165 turtles admitted per annum, costing approximately $AUD1,000 per animal admitted if you average total money spent on marine turtle rehabilitation per animal admitted. Over the 18 years of this study, 1,173 of these turtles were released, equaling approximately 65 turtles per year at an estimated cost of $AUD2,583 per animal released from rehabilitation if you average total money spent on marine turtle rehabilitation per animal released. Over the 18 years of this study, 101 of these released turtles were recaptured, equaling an average of 5.6 turtles per year and costing on average $AUD29,464 to rehabilitate each animal that is caught again. Of all of these animals admitted across all rehabilitation facilities for marine turtles, rehabilitated, released and recaptured, only 24 turtles were recaptured as functioning healthy members of the wild population, equaling approximately 1.3 turtles per year at a cost of $AUD123, 750 to return a single animal to the functional population.When analyzing the costs for animals which were not successful during rehabilitation it is estimated that approximately $AUD1650 is spent per turtle which is either euthanized or dies while in care.This investigation found that different causes of stranding influenced the survival for individuals, in terms of length of time in care, and survival of rehabilitation and post rehabilitation success. This provides rehabilitation centres with important information about resource outlay, particularly if success rates are poor (approximately 8.6%).R2 = 0.6377); (ii) a species (loggerhead and green turtles (77.4%)) prevalence; (iii) a seasonal effect on different age classes, with most overall strandings occurring between August and November (47%); and (iv) stranding hotspots  persisting throughout the study timeframe. These hotspots correspond to major freshwater discharge points as well as highly developed/populated areas.When analysing stranding data for Queensland between 1996 and 2013, Green turtles were the most frequent species and immature turtles were the most frequent age class sent to rehabilitation both by numbers and proportion, most likely because green turtles represent the largest proportion of the Queensland marine turtle populations and small immature turtles are the largest cohort of this population . FurtherIncreased morbidity and mortality is often associated with the perils of anthropogenic interactions. Green turtle stranding increased at a rate of 9.9% per annum (pa) over the study period. However, during a similar timeframe, the southern Great Barrier Reef green turtle population increased at a rate of approximately 10.6% pa . As wellTrichechus manatus latirostris) in high use areas  . As the se areas .For a short period between 2005 and 2009, euthanasia rates increased across rehabilitation centers in Queensland . As therDuring rehabilitation, over 25% of turtles die unassisted during the first week of treatment , suggestSome of the results found in this study differ from that of Similar rehabilitation results were obtained by It is difficult to assess the true success of rehabilitation without following each individual. There have been few studies investigating the ability of turtles to readapt to the wild after rehabilitation . The twoThe number of turtles reported stranded during this study represent approximately >0.001% ((9641 turtles/18 years)/641262) of the suspected benthic southern Great Barrier Reef population . DespiteBased on statewide recapture data and the cost of rehabilitation at the three main rehabilitation centres in Queensland , only 1.3 turtles are successfully returned to the functional population each year at a cost of $AUS123,750 an animal. Even though these costs should only be viewed as indicative estimates as opposed to a true calculation per known animal released, the pattern indicates this may not be economical and these high costs and low numbers are likely not contributing to conserving a population . HoweverThere is no question rehabilitation plays an important role in the care of individual turtles by reducing suffering and treatment of certain conditions. Part of the rehabilitation process can include euthanasia as a treatment option to prevent individual suffering and can add value to research through appropriate post-mortem investigations. Rehabilitation also provides a valuable vehicle for public education and conservation messages which, in turn, increase public awareness and hopefully reduce anthropogenic causes of stranding . This raGiven the prevalence of: (i) certain cohorts; (ii) seasonality; (iii) certain causes of stranding; and (iv) higher numbers of strandings at four locations along the Queensland coastline (hotspots), it may be more appropriate to direct rehabilitation efforts to events of higher demand. For example, this could mean creating MASH -like response centers that target their care to immature turtles that present at the end of the winter period (August–September)—to treat/evaluate boat strike and unknown causes, within the recognized stranding ‘hotspots’. Focused triage and treatment may represent a significant cost saving to rehabilitation centers throughout Australia. As 22% of animals which are admitted to rehabilitation reach an outcome within 7 days, the creation of such response centers, will allow turtles with obvious disorders which only need short term immediate care to be treated and released, freeing up resources and space in rehabilitation centers for turtles which require more in-depth/long-term care.Despite the costs involved, rehabilitation continues to be a tool for conservation because it provides a platform to educate members of the public about threats to marine turtle survival . It has 10.7717/peerj.3132/supp-1Table S1Click here for additional data file.10.7717/peerj.3132/supp-2Table S2Click here for additional data file.10.7717/peerj.3132/supp-3Data S1Click here for additional data file.