Additional file 3: of Characterisation of microbial communities within aggressive prostate cancer tissues

Complete 16 rRNA V2-V3 region taxa summary. (DOCX 1291 kb

The translation efficiency of the HIF1α 5′UTR-luciferase reporter in prostate cancer cells.

<p>(A) <i>Firefly</i> and <i>Renilla</i> luciferase activities in prostate cancer cells following transfection of a HIF1α 5′UTR-luciferase construct and the pTK-Renilla control reporter vector were determined using a dual luciferase assay. (B) Real-time PCR (RT-PCR) analysis of luciferase mRNA in PC cells transfected with the HIF1α 5′UTR-luciferase construct. Following transfection, RNA was isolated, and luciferase mRNA expression detected by real time RT-PCR and normalized by 18S mRNA expression. (C) Translational efficiency represents the ratio of <i>Firefly</i>/<i>Renilla</i> luciferase activity, divided by the relative luciferase mRNA concentration in PC cells. The translational efficiency of luciferase mRNA driven by the 5′UTR region of HIF1α in PC3 cells is higher than in LNCaP cells. Values are the mean ± SEM of at least three separate experiments. *, P<0.05 versus treated LNCaP cells.</p

Basal HIF1α protein expression, proliferation rates and migration/invasion rates in human PC cell lines. (

<p>A) Basal HIF1α protein concentrations in the human PC cell lines LNCaP, DU145 and PC3 under normoxic conditions were analyzed by Western blot. (B) Proliferation was assayed by cell counting after 24 and 48 hours. (C) Migration/invasion rates were measured by Transwell assays at 24 hours. Values in (A) and (C) are expressed as the fold increase compared to LNCaP cells, while the values in (B) are expressed as a percentage of the time 0 value. All values are the mean ± SEM of at least three separate treatments. (D) Survival rates of PC cells exposed to cytotoxic conditions. The survival of PC3 cells (which have higher basal HIF1α protein) when exposed to oxidative stress with hydrogen peroxide (H₂O₂) or chemotoxicity with 5-fluorouracil (5-FU) was compared to the survival of LNCaP cells (which have lower HIF1α expression). Survival was assessed by counting cell numbers at 24 hours. Values are expressed as a percentage of the untreated control and are the mean ± SEM of at least three separate treatments. #, P<0.05 versus treated LNCaP cells.</p

Patient characteristics for the groups with Gleason score ≤7 or >7 and differences in HIF1α expression between the groups.

<p>The group with Gleason score ≤7 was comprised of Gleason score <6 (6), 6 (28) and 7 (4) and the group with Gleason score >7 was comprised of scores 8 (8), 9 (52) and 10 (2).</p>†<p>There were 17 patients with missing data for tumor stage in the group with Gleason score >7 and 16 of these patients were HIF1α positive.</p>¥<p>There was no significant association with HIF1α expression and Gleason scores when analyzed using Pearson’s Chi-squared test or Fisher’s exact test using two-by-two tables.</p>*<p>Pre-interventional PSA was defined as PSA immediately prior to obtaining the tissue sample.</p

Knockdown of HIF1α expression in PC3 cells reduced both survival after cytotoxic treatments and migration rate.

<p>(A) HIF1α concentrations were reduced in 2 separate clones of PC3 cells following stable expression of HIF1α shRNA as assessed by Western blot. Values are the mean ± SEM of at least three separate experiments and are expressed as a percentage of wild-type PC3 cells. *, P<0.05 versus wild-type PC3 cells. (B) The survival of PC3 cells after exposure to oxidative stress (hydrogen peroxide (H₂O₂)) or chemotoxicity (5-fluorouracil (5-FU) for 24 hours was reduced following HIF1α knockdown compared to scrambled control vector-transfected PC3 cells. Values are the mean ± SEM of at least three separate experiments and are expressed as a percentage of untreated scrambled control vector-transfected PC3 cells. #, P<0.05 versus control. (C) HIF1α protein expression in PC3 cells transfected with control shRNA after treatment with 1% O₂, 300 µM CoCl₂, 100 µM H₂O₂, and 15 µM 5-FU. Cell lysates were electrophoresed on SDS-polyacrylamide gels and blotted with HIF1α antibody. GAPDH expression was used as loading control. The Western blots shown are representative of at least three separate experiments. Band densities were determined by densitometric analysis of HIF1α/GAPDH and are presented relative to the value for untreated cells. Data represent mean ± SEM; * p<0.05 vs. untreated PC3 cells. (D) Rates of migration/invasion in the HIF1α knockdown PC3 cells were reduced compared to the scrambled control vector-transfected PC3 cells as assessed by Transwell assay. Values are the mean ± SEM of at least three separate experiments and are expressed as a percentage of untreated scrambled control vector transfected PC3 cells. *, P<0.05 versus control. (E) Induction of HIF1α in LNCaP cells by hypoxia (dark grey bars) or by cobalt chloride (light grey bars) increased survival after exposure to oxidative stress with H₂O₂ or chemotoxicity with 5-FU for 24 hours when compared to control LNCaP cells (black bars). Values are the mean ± SEM of at least three separate treatments and are expressed as a percentage of the untreated LNCaP control. #, P<0.05 versus treated LNCaP cells. *, P<0.05 versus LNCaP cells treated with 1% O₂ and 5-FU. (F) HIF1α protein expression in LNCaP cells treated with 1% O₂ and 300 µM CoCl₂ in combination with either 100 µM H₂O₂ or 15 µM 5-FU. Cell lysates were electrophoresed on SDS-polyacrylamide gels and blotted with HIF1α antibody. GAPDH expression was used as loading control. The Western blots shown are representative of at least three separate experiments. Band densities were determined by densitometric analysis of HIF1α/GAPDH and are presented relative to the value for normoxic cells undergoing the same treatment. Data represent mean ± SEM; * p<0.05 vs. untreated control, 100 µM H₂O₂ or 15 µM 5-FU treated LNCaP cells.</p

Univariate and Multivariate Cox Regression analysis of the development of metastatic PC from the time of surgery and CRPC, prostate cancer specific death after starting androgen deprivation therapy.

a<p>13 patients excluded due to incomplete metastasis related data.</p>†<p>Cox regression with Firth’s penalized maximum likelihood method. CI denotes confidence interval.</p>‡<p>This group served as the reference group in the Cox regression analysis.</p>*<p>Pre-interventional PSA was defined as PSA immediately prior to obtaining the tissue sample.</p

Machine learning to support social media empowered patients in cancer care and cancer treatment decisions

<div><p>Background</p><p>A primary variant of social media, online support groups (OSG) extend beyond the standard definition to incorporate a dimension of advice, support and guidance for patients. OSG are complementary, yet significant adjunct to patient journeys. Machine learning and natural language processing techniques can be applied to these large volumes of unstructured text discussions accumulated in OSG for intelligent extraction of patient-reported demographics, behaviours, decisions, treatment, side effects and expressions of emotions. New insights from the fusion and synthesis of such diverse patient-reported information, as expressed throughout the patient journey from diagnosis to treatment and recovery, can contribute towards informed decision-making on personalized healthcare delivery and the development of healthcare policy guidelines.</p><p>Methods and findings</p><p>We have designed and developed an artificial intelligence based analytics framework using machine learning and natural language processing techniques for intelligent analysis and automated aggregation of patient information and interaction trajectories in online support groups. Alongside the social interactions aspect, patient behaviours, decisions, demographics, clinical factors, emotions, as subsequently expressed over time, are extracted and analysed. More specifically, we utilised this platform to investigate the impact of online social influences on the intimate decision scenario of selecting a treatment type, recovery after treatment, side effects and emotions expressed over time, using prostate cancer as a model. Results manifest the three major decision-making behaviours among patients, Paternalistic group, Autonomous group and Shared group. Furthermore, each group demonstrated diverse behaviours in post-decision discussions on clinical outcomes, advice and expressions of emotion during the twelve months following treatment. Over time, the transition of patients from information and emotional support seeking behaviours to providers of information and emotional support to other patients was also observed.</p><p>Conclusions</p><p>Findings from this study are a rigorous indication of the expectations of social media empowered patients, their potential for individualised decision-making, clinical and emotional needs. The increasing popularity of OSG further confirms that it is timely for clinicians to consider patient voices as expressed in OSG. We have successfully demonstrated that the proposed platform can be utilised to investigate, analyse and derive actionable insights from patient-reported information on prostate cancer, in support of patient focused healthcare delivery. The platform can be extended and applied just as effectively to any other medical condition.</p></div

Cobalt and zinc induce HIF1α and HIF2α but not HIF3α.

<p>(A) Western blots revealed that ZnCl₂ induces HIF1α protein in the human renal cancer cell line ACHN and the immortalized human renal tubular cell line HK-2 in a dose-dependent manner. The expression of HIF1α, HIF2α, HIF3α, phosphorylated-AKT and phosphorylated ERK1/2 proteins in ACHN (B, C) and HK-2 (D, E) cells after treatment with 150μM CoCl₂ or 50μM ZnCl₂ for 4 or 24 hours was measured by Western blot (B, D) and analysed by densitometry (C, E). Protein expression was normalized to glyceraldeyde-3-phosphate dehydrogenase (GAPDH), and expressed as the fold increase relative to untreated control cells. Data are mean ± SEM from at least three independent experiments. *, P<0.05 vs. control, ^#, P<0.05 vs. 150μM CoCl₂.</p

Zinc preconditioning protects against renal ischemia reperfusion injury in rats.

<p>(A) Diagram showing the experimental protocol. (B) Serum creatinine concentrations (mean ± SEM (μmol/L)) in rats treated with saline (control) (●), 30 mg/kg cobalt (■), 5 mg/kg ZnCl₂ (▲), 10 mg/kg ZnCl₂ (▼) or 30 mg/kg ZnCl₂ (◆). The rise in serum creatinine was less in rats preconditioned with 10mg/kg ZnCl₂ than in the saline control group. (C) Serum urea concentrations (mean ± SEM (mmol/L)). The rise in serum urea was less in rats preconditioned with 10 mg/kg ZnCl₂ than in the saline control group. (D) ZnCl₂ preconditioning improves the health of rats after IRI. The slope (-0.51) of the line of regression for weight loss in the rats treated with 10 mg/kg ZnCl₂ was less than the slope for rats treated with 30 mg/kg cobalt (-1.32) or saline (-1.15). *p<0.05 Vs saline treated control. On repeated measures ANOVA, including days one to seven, there was a statistically significant difference between- 10 mg/kg ZnCl₂ Vs Saline control (p = 0.038).</p

Preconditioning with CoCl₂ is more effective than intermittent clamping (IC).

<p>(A) Experimental design for the <i>in vivo</i> study comparing the ability of different preconditioning techniques to protect against IRI in rats. (B) Animal sickness scores in rats treated with saline (control) (●), 30 mg/kg CoCl₂ (■), IC (⬢) or IC + CoCl₂ (▲). (C) Serum creatinine concentrations (mean ± SEM (µmol/L)). The rise in serum creatinine was less in rats preconditioned with 30 mg/kg CoCl₂ than in the saline control group. (D) Serum urea concentrations (mean ± SEM (mmol/L)). *p<0.05 Vs saline treated control.</p