Effects of intentionally treated water on the growth of mesenchymal stem cells: An exploratory study.

OBJECTIVE: This study explored if human primary mesenchymal stem cells (MSCs), derived from two donors and cultivated in a medium made with intentionally treated water, would exhibit more growth and pluripotency than MSCs from the same source but grown in untreated (control) water. DESIGN: To create the treated water, three Buddhist monks directed their attention toward commercially bottled water while holding the intention that the water would enhance the growth of MSCs. Under double-blind conditions, cell culture growth mediums were prepared with the treated and untreated water, which was in turn used to grow the primary MSCs. Primary cells obtained from two donors were designated as Cells #1 and Cells #2. The prediction was that treated water would result in increased cell proliferation, that more cells would enter the cell cycle growth phase, and that there would be increased expression of genes (NANOG, OCT4 and SOX2) associated with improved cell growth and decreased expression of genes (p16, p21, and p53) associated with a decline in cell growth. The improved growth hypothesis was directional, thus one-tailed p-values were used to evaluate the results. RESULTS: Proliferation averaged across Cells #1 and #2 showed overall increased growth in treated as compared to control water (p = 0.0008). Cells #1 and #2 considered separately had differences in the same direction but only Cells #2 showed a significant difference on day 6 (p = 0.01). For cell cycle, there was a significantly greater percentage of Cells #2 in the S interphase with treated vs. control water (p = 0.04). For the gene expression analysis, when considering the average across the two donor cells, only the NANOG gene expression was in the predicted direction (p = 0.01); by contrast, the p16 gene expression was significantly opposite to the predicted direction (p = 0.005, one-tailed, post-hoc). For Cells #1 considered separately, no differences were significant except for p16, which resulted in an effect opposite to the predicted outcome (p = 0.05). For Cells #2, three genes were significantly in the predicted directions: NANOG (p = 0.0008), OCT4 (p = 0.005), and P53 (p = 0.05); p16 was significantly opposite to the prediction (p = 0.001). CONCLUSION: Intentionally treated water appeared to have some biological effects on the growth, pluripotency and senescence of human MSCs. This was especially the case in one of the two donor cells tested, but the effects were not consistently in the predicted direction. As an exploratory study, caution is warranted in interpreting these outcomes, and adjustment for multiple testing would likely reduce some of the weaker effects to nonsignificant. But given the double-blind protocol, as well as several more significant outcomes in the predicted directions, further research is warranted.

Statins regulate interleukin-1ß-induced RANKL and osteoprotegerin production by human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Three-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase competitive inhibitors, or 'statins', are widely used for lowering cholesterol and thereby reducing the risk of a heart attack. Recent data suggest that statins influence metabolic bone activity by their actions on three molecules: RANKL; RANK; and osteoprotegerin (OPG), the soluble decoy receptor for RANKL. The purpose of this study was to evaluate OPG and RANKL production in resting and interleukin-1ß (IL-1ß)-activated human gingival fibroblasts (HGFs), and to determine the effect of statins on their production. MATERIAL AND METHODS: Fibroblasts were pre-incubated with atorvastatin or simvastatin for 24h in serum-free medium, and then incubated with IL-1ß for 6d. The concentration of OPG or RANKL in culture supernatants was measured by specific ELISA. Data were analyzed using analysis of variance and Scheffe's F procedure for post hoc comparison. RESULTS: IL-1ß (1×10(-8) m) stimulated a significant increase in the production of OPG on days 1, 3 and 6. There was a trend towards an increase in RANKL production as a result of stimulation with IL-1ß. Both statins, at multiple concentrations, significantly increased the constitutive RANKL/OPG ratio. Only atorvastatin at the highest concentration (5×10(-6) m) significantly increased the IL-1ß-stimulated RANKL/OPG ratio. CONCLUSION: IL-1ß significantly increased OPG production by HGFs. The statins differed minimally in their effects on OPG and RANKL production by resting and IL-1ß-activated HGFs. Both statins increased constitutive RANKL/OPG ratios, but generally not IL-1ß-stimulated ratios. Thus, statins may influence the production of RANKL and OPG by HGFs to favor bone catabolism, under noninflammatory conditions.

Effects of intentionally treated water and seeds on the growth of Arabidopsis thaliana.

OBJECTIVE: . A previously reported experiment indicated that Arabidopsis thaliana seeds with cryptochrome mutation His-CRY2 showed more robust photomorphogenic growth when hydrated with intentionally treated water as compared to untreated water. The present study attempted to replicate that outcome, adding a condition where the seeds were also intentionally treated. Arabidopsis seeds were used because they contain a photosensitive flavoprotein called cryptochrome (CRY). CRY has been proposed as a possible "transducer" of intention in living systems because it is thought to have quantum biological properties, and as such, it might potentially be sensitive to quantum observer effects. DESIGN: Three Buddhist monks directed their attention toward commercially bottled water and Arabidopsis seeds while holding the intention to improve the growth of the plant. As a control condition, no attention was directed at water or seeds from the same sources. Under double-blinded conditions, treated and untreated seeds were placed in an incubator, hydrated with treated or untreated water, and exposed to either continuous blue light or blue plus far-red light. The seed germination process was repeated three times, each time using new seeds. A 2 × 2 × 2 ANOVA, with water, seeds, and light as factors, was used to analyze the results. RESULTS: . Treated water was associated with enhanced photomorphogenic growth, as reflected by a shorter hypocotyl length (p = 0.04) and greater amounts of chlorophyll (p = 0.0005) and anthocyanin (p = 2 × 10-6). Treated seeds resulted in greater amounts of chlorophyll (p = 0.04), but also a longer hypocotyl (p = 0.0004) and less anthocyanin (p = 0.01). Plants exposed to blue plus far-red light were constantly more robust than plants grown under blue light, regardless of the type of water or seed (p < 10-10). CONCLUSION: . Intentionally treated water improved the growth of the His-CRY2 variant of Arabidopsis, confirming results of an earlier experiment. Enhanced growth associated with exposure to blue plus far-red light also confirmed to known effects. A more complex relationship was observed with treated seeds. Further research is required to understand the latter outcome, as it may provide clues about the underlying mechanisms of intentional influences.

Effects of Intentionally Treated Water on Growth of Arabidopsis thaliana Seeds with Cryptochrome Mutations.

OBJECTIVE: A previous experiment suggested that consumption of intentionally treated tea influenced subjective mood under double-blind, controlled conditions. To investigate that effect objectively, again under double-blind, controlled conditions, we studied whether Arabidopsis thaliana seeds hydrated with intentionally treated vs. untreated water would show differences in hypocotyl length, anthocyanin, and chlorophyll. DESIGN: Three Buddhist monks focused their intention on commercially bottled water with the goal of improving the growth of seeds; bottled water from the same source served as an untreated control. Seeds with the following three variations of cryptochrome (CRY) were used: the wild type Arabidopsis (Columbia-4), a gain-of-function mutation (His-CRY2), and a loss-of function mutation (cry1/2), where "gain" and "loss" refer to enhanced and reduced sensitivity to blue light, respectively. Seeds were hydrated with treated or untreated water under blinded conditions, and then placed in random positions in an incubator. The germination process was repeated three times in each experiment, each time using new seeds, and then the entire experiment was repeated four times. RESULTS: Data combined across the four experiments showed a significant decrease in hypocotyl length in the His-CRY2 seedlings (treated mean 1.31 ± 0.01mm, untreated mean 1.43 ± 0.01mm, P < 10-13), a significant increase in anthocyanin with all three forms of cry, particularly His-CRY2 (treated mean 17.0 ± 0.31mg, untreated mean 14.5 ± 0.31mg, P < 10-4), and a modest increase in chlorophyll in His-CRY2 (treated mean 247.6 ± 5.63mg, untreated mean 230.6 ± 5.63mg, P = .05). These outcomes conformed to the monks' intentions because a decrease in hypocotyl length and increase in anthocyanin and chlorophyll are associated with enhanced photomorphogenic growth. These experiments suggest that the His-CRY2 mutation of Arabidopsis may be an especially robust "detector" of intention.

Extracellular Matrix Hydrogel Promotes Tissue Remodeling, Arteriogenesis, and Perfusion in a Rat Hindlimb Ischemia Model.

OBJECTIVE: This study aimed to examine acellular extracellular matrix based hydrogels as potential therapies for treating peripheral artery disease (PAD). We tested the efficacy of using a tissue specific injectable hydrogel, derived from decellularized porcine skeletal muscle (SKM), compared to a new human umbilical cord derived matrix (hUC) hydrogel, which could have greater potential for tissue regeneration because of its young tissue source age. BACKGROUND: The prevalence of PAD is increasing and can lead to critical limb ischemia (CLI) with potential limb amputation. Currently there are no therapies for PAD that effectively treat all of the underlying pathologies, including reduced tissue perfusion and muscle atrophy. METHODS: In a rodent hindlimb ischemia model both hydrogels were injected 1-week post-surgery and perfusion was regularly monitored with laser speckle contrast analysis (LASCA) to 35 days post-injection. Histology and immunohistochemistry were used to assess neovascularization and muscle health. Whole transcriptome analysis was further conducted on SKM injected animals on 3 and 10 days post-injection. RESULTS: Significant improvements in hindlimb tissue perfusion and perfusion kinetics were observed with both biomaterials. End point histology indicated this was a result of arteriogenesis, rather than angiogenesis, and that the materials were biocompatible. Skeletal muscle fiber morphology analysis indicated that the muscle treated with the tissue specific, SKM hydrogel more closely matched healthy tissue morphology. Short term histology also indicated arteriogenesis rather than angiogenesis, as well as improved recruitment of skeletal muscle progenitors. Whole transcriptome analysis indicated that the SKM hydrogel caused a shift in the inflammatory response, decreased cell death, and increased blood vessel and muscle development. CONCLUSION: These results show the efficacy of an injectable ECM hydrogel alone as a potential therapy for treating patients with PAD. Our results indicate that the SKM hydrogel improved functional outcomes through stimulation of arteriogenesis and muscle progenitor cell recruitment.

Gut feelings, intuition, and emotions: an exploratory study.

OBJECTIVE: Investigate whether the gut feelings of one person, as measured with an electrogastrogram (EGG), respond to the emotions of a distant person. DESIGN: In a double blind protocol, EGG activity was recorded in an individual relaxing in a heavily shielded chamber while, at a distance, a second person periodically viewed the live video image of the first person along with stimuli designed to evoke positive, negative, calming, or neutral emotions. SUBJECTS: Twenty-six (26) pairs of healthy adult volunteers. OUTCOME MEASURES: EGG maximum values recorded while the distant person was exposed to emotional stimuli were compared to similar values recorded during exposure to neutral stimuli. RESULTS: EGG maximums were significantly larger on average when the distant person was experiencing positive (p = 0.006) and negative (p = 0.0009) emotions, as compared to neutral emotions. Nonparametric bootstrap procedures were employed to evaluate these differences, and the results survive correction for multiple analyses. CONCLUSIONS: EGG activity increases in response to the emotions of a distant person, beyond the influence of ordinary sensory interactions. Relationships commonly reported between gut feelings and intuitive hunches may share a common, poorly understood, perceptive origin.

Human breast cancer invasion and aggression correlates with ECM stiffening and immune cell infiltration.

Tumors are stiff and data suggest that the extracellular matrix stiffening that correlates with experimental mammary malignancy drives tumor invasion and metastasis. Nevertheless, the relationship between tissue and extracellular matrix stiffness and human breast cancer progression and aggression remains unclear. We undertook a biophysical and biochemical assessment of stromal-epithelial interactions in noninvasive, invasive and normal adjacent human breast tissue and in breast cancers of increasingly aggressive subtype. Our analysis revealed that human breast cancer transformation is accompanied by an incremental increase in collagen deposition and a progressive linearization and thickening of interstitial collagen. The linearization of collagen was visualized as an overall increase in tissue birefringence and was most striking at the invasive front of the tumor where the stiffness of the stroma and cellular mechanosignaling were the highest. Amongst breast cancer subtypes we found that the stroma at the invasive region of the more aggressive Basal-like and Her2 tumor subtypes was the most heterogeneous and the stiffest when compared to the less aggressive luminal A and B subtypes. Intriguingly, we quantified the greatest number of infiltrating macrophages and the highest level of TGF beta signaling within the cells at the invasive front. We also established that stroma stiffness and the level of cellular TGF beta signaling positively correlated with each other and with the number of infiltrating tumor-activated macrophages, which was highest in the more aggressive tumor subtypes. These findings indicate that human breast cancer progression and aggression, collagen linearization and stromal stiffening are linked and implicate tissue inflammation and TGF beta.

Event-related electroencephalographic correlations between isolated human subjects.

OBJECTIVE: To examine electroencephalograms (EEG) in pairs of people to see if event-related potentials evoked in one person's brain are correlated with concurrent responses in the brain of a distant, isolated person. DESIGN: Simultaneously record EEGs using independent physiologic monitoring systems. One person relaxes in a double steel-walled, electromagnetically and acoustically shielded room while a second, located in a dimly lit room 20 meters away, is stimulated at random times by the live video image of the first person. SUBJECTS: Thirteen (13) pairs of volunteers. Eleven (11) pairs of adult friends and 2 mother-daughter pairs. OUTCOME MEASURES: Epochs of interest were the moments of stimulus onset and offset, +/- 5 seconds, in both participants' EEGs. A positive correlation was postulated to appear between the ensemble variance of the stimulated subjects' EEGs versus an identical measure in the nonstimulated subjects. Control data using the same equipment and test conditions, but without humans present, was collected to check for equipment and analytical artifacts. Nonparametric bootstrap methods were used to assess statistical significance of the observed correlations. RESULTS: The control test resulted in a correlation of r =-0.03, p = 0.61; the experimental test resulted in r = 0.20, p = 0.0005. Three (3) of the 13 pairs of participants showed independently significant correlations. Examination of the stimulated subjects' event-related potentials showed that the stronger their responses, the larger the corresponding responses in the nonstimulated subjects (p = 0.0008). CONCLUSION: Under certain conditions, the EEG of a sensorially isolated human subject can become correlated with event-related potentials in a distant person's EEG. This suggests the presence of an unknown form of energetic or informational interaction.

Electrocortical activity prior to unpredictable stimuli in meditators and nonmeditators.

Advanced meditators occasionally report experiences of timelessness, or states of awareness that seem to transcend the usual boundaries of the subjective present. This type of experience was investigated in eight experienced meditators and eight matched controls by measuring 32 channels of EEG before, during, and after exposure to unpredictable light and sound stimuli. The experiment postulated that if some aspect of consciousness extends beyond the present moment, then prestimulus electrocortical signals should differ depending on stimuli that were about to be selected by a truly random process, and that if such experiences were catalyzed through meditation practice, then prestimulus differences should be more apparent in meditators than in nonmeditators. Each of the 32 EEG channels was baseline adjusted on each trial by the electrical potential averaged between two- and one-second prestimulus, then for each channel the average potential was determined from one-second prestimulus to stimulus onset. The resulting means across subjects in each group were compared by stimulus type using randomized permutation procedures and corrected for multiple comparisons. Within the control group, no EEG channels showed significant prestimulus differences between light versus sound stimulus conditions, but within the meditator group five of 32 channels resulted in significant differences (P < .05, two tailed). Comparisons between control and meditator groups showed significant prestimulus differences prior to audio tone stimuli in 14 of 32 channels (P < .05, two tailed, of which eight channels were P < .005, two tailed). This outcome successfully replicates effects reported in earlier experiments, suggesting that sometimes the subjective sense of awareness extending into the future may be ontologically accurate.

Chronic (1-year) oral toxicity study of erythritol in dogs.

The chronic oral toxicity of erythritol was examined by feeding erythritol at dietary levels of 0 (controls), 2, 5, or 10% to groups of four male and four female dogs for 53 weeks. Erythritol was well tolerated at all dose levels without evidence of diarrhea. Water consumption was slightly higher in the high-dose group than in controls. Body weights and weight gains were not affected by treatment. There were no clinically relevant changes in hematological or clinicochemical parameters attributable to treatment. In particular, plasma electrolyte concentrations remained unaffected. Evaluation of a number of urinary parameters (including electrolytes and renal enzymes) was hampered by widely varying urine volumes among individual dogs; however, the available data did not indicate treatment-related effects on the urinary excretion of electrolytes (K+, Na+, Mg2+, and Pi) or enzymes (gamma-glutamyltranspeptidase, N-acetyl glucosaminidase, and lactate dehydrogenase). Quantitation of erythritol in the urine demonstrated that 50 to 80% of the ingested dose was absorbed and excreted in the urine. Analysis of terminal organ weights did not reveal treatment-related differences. No histopathological changes attributable to treatment were observed in the kidneys or in any other organ or tissue examined. It was concluded that daily erythritol consumption of up to 3.5 g/kg body wt was well tolerated by dogs.