Alterations in folate-dependent one-carbon metabolism as colon cell transition from normal to cancerous.

Folate-dependent one-carbon cycle metabolism (FOCM) plays a critical role in maintaining genomic stability through regulating DNA biosynthesis, repair and methylation. Folate metabolites as well as other metabolites in the FOCM are hypothesized to be altered when cells transition from normal to cancerous state. Using cells at different stages in their development into colorectal cancer, the FOCM metabolites were profiled as an effort to phenotype the cells, and the metabolite levels were compared to the expressions of related genes. Here, we investigate whether there is a correlation between the metabolite levels, DNA methylation levels and the expression of the related genes that drive the levels of these metabolites. Using CRL1459, APC10.1, HCT116 and Caco-2, we show for the first time that FOCM metabolites correlate with the gene expression patterns. These differences follow a trend that may facilitate distinguishing colon cells at the different stages as they transition into cancerous state. The folate distribution and methionine levels were found to be key in determining the staging of the colon cells in CRC development. Also, expression of CBS, MTRR and MAT genes may facilitate distinguishing between untransformed and transformed colon cells.

BCL11A enhancer haplotypes and fetal hemoglobin in sickle cell anemia.

BACKGROUND: Fetal hemoglobin (HbF) levels in sickle cell anemia patients vary. We genotyped polymorphisms in the erythroid-specific enhancer of BCL11A to see if they might account for the very high HbF associated with the Arab-Indian (AI) haplotype and Benin haplotype of sickle cell anemia. METHODS AND RESULTS: Six BCL112A enhancer SNPs and their haplotypes were studied in Saudi Arabs from the Eastern Province and Indian patients with AI haplotype (HbF ~20%), African Americans (HbF ~7%), and Saudi Arabs from the Southwestern Province (HbF ~12%). Four SNPs (rs1427407, rs6706648, rs6738440, and rs7606173) and their haplotypes were consistently associated with HbF levels. The distributions of haplotypes differ in the 3 cohorts but not their genetic effects: the haplotype TCAG was associated with the lowest HbF level and the haplotype GTAC was associated with the highest HbF level and differences in HbF levels between carriers of these haplotypes in all cohorts were approximately 6%. CONCLUSIONS: Common HbF BCL11A enhancer haplotypes in patients with African origin and AI sickle cell anemia have similar effects on HbF but they do not explain their differences in HbF.

Lipoprotein lipase deficiency leads to α-synuclein aggregation and ubiquitin C-terminal hydrolase L1 reduction.

We have previously reported that presynaptic dysfunction and cognitive decline have been found in lipoprotein lipase (LPL) deficient mice, but the mechanism remains to be elucidated. Accumulating evidence supported that α-synuclein (α-syn) and ubiquitin C-terminal hydrolase L1 (UCHL1) are required for normal synaptic and cognitive function. In this study, we found that α-syn aggregated and the expression of UCHL1 decreased in the brain of LPL deficient mice. Reduction of UCHL1 was resulted from nuclear retention of DNA cytosine-5-methyltransferase 1 in LPL knockout mice. Reverse changes were found in cultured cells overexpressing LPL. Furthermore, deficiency of LPL increased ubiquitination of α-syn. These results indicated that aggregation of α-syn and reduction of UCHL1 expression in LPL-deficient mice may affect synaptic function.

Impaired synaptic vesicle recycling contributes to presynaptic dysfunction in lipoprotein lipase-deficient mice.

Lipoprotein lipase (LPL) is expressed at high levels in hippocampal neurons, although its function is unclear. We previously reported that LPL-deficient mice have learning and memory impairment and fewer synaptic vesicles in hippocampal neurons, but properties of synaptic activity in LPL-deficient neurons remain unexplored. In this study, we found reduced frequency of miniature excitatory postsynaptic currents (mEPSCs) and readily releasable pool (RRP) size in LPL-deficient neurons, which led to presynaptic dysfunction and plasticity impairment without altering postsynaptic activity. We demonstrated that synaptic vesicle recycling, which is known to play an important role in maintaining the RRP size in active synapses, is impaired in LPL-deficient neurons. Moreover, lipid assay revealed deficient docosahexaenoic acid (DHA) and arachidonic acid (AA) in the hippocampus of LPL-deficient mice; exogenous DHA or AA supplement partially restored synaptic vesicle recycling capability. These results suggest that impaired synaptic vesicle recycling results from deficient DHA and AA and contributes to the presynaptic dysfunction and plasticity impairment in LPL-deficient neurons.

A double-blind, rct testing beneficial modulation of BDNF in middle-aged, life style-stressed subjects: a clue to brain protection?

INTRODUCTION: The aim of this prospective study was to see whether LD-1227, a quality-controlled marine nutraceuticals shown to protect experimental stress-induced hyppocampal degeneration, could beneficially modulate BDNF, as measured in the serum, in otherwise healthy but work-stressed individuals. MATERIALS AND METHODS: Forty-eight men and women between the ages of 38 and 62 reporting high-demanding work activity but with an overall positive attitude towards their personal life were recruited. Subjects were divided in two group (24 patients each) and blindly supplemented for 2 month with: a) LD-1227 400mg or b) placebo. A third group of healthy non-stressed subjects was used as well. Blood samples were taken before and after the supplementation period. Unstimulated saliva was collected and tested for amylase while serum levels were used to measure BDNF. State Trait Anxiety Inventory (STAI), Pittsburgh Sleep Quality Index (PSQI) and psychological well-being assessment (PSWB) were measured too. Patients with Val66Met functional polymorphism of BDNF excluded those given their reported association with an impaired release of BDNF. RESULTS: RESULTS showed that, as compared to healthy, non-stressed individuals, stressed ones has a trend decrease of BDNF and this was significantly increased by LD 12-1227 supplementation and the same inverse phenomenon occurred to salivary amylase (p<0.05). No change was noted in the PSQI score but, either STAI or PSWB tests scored better in LD-1227 supplemented subjects. CONCLUSION: The present data suggest that LD-1227 is beneficially affecting neuromodulation and related symptoms during common stressful life conditions and may have the potential as tools in a neuroprotective clinical strategy.

Effective properties of a sturgeon-based bioactive compound on stress-induced hippocampal degeneration and on in vitro neurogenesis.

The aim of this study is to test the activity of a marine bioactive compound containing high-purity caviar-derived DNA, collagen elastin and protein extracts from sturgeon (LD-1227, Caviarlieri, Laboratoires Dom, Switzerland) to exert neuroprotective properties in an experimental setting while also being potential triggers of neurogenesis in a separate in vitro study. Supplementation with high-DHA mixture of LD-1227 was applied for 30 days to stress model rats. Both supplementations significantly mitigated the histological brain damage when analyzing hippocampal subregions and corticosterone level. However, LD-1227 was most significantly efficient in preventing SOD, Catalase and ascorbic acid decrease in brain tissue. Both supplementations stimulated neurogenesis in vitro and neuron markers in particular but og olygodendrocyte markers and glia increased only in LD-1227-enriched medium. Taken together, these data suggest that LD-1227 is able to significantly protect the brain structure redox system to higher degree than DHA. Moreover, from in vitro study it appears that marine bioactive compound, through it wide array of small unsaturated fatty acids, phospholipids and neurotransmitter precursors, is likely to influence neuronal and glial lineage to act differently from a DHA-rich mixture.

Advantage of carbonate-versus citrate-based alkalinization on bone metabolism in moderately exercising aged male rats fed an acidogenic diet.

This study aims to determine the effects of different alkaline supplementations on high protein diet-induced abnormalities affecting bone metabolism in rats which were also undergoing physical exercise of moderate intensity. Sixty elderly Sprague-Dawley rats were randomly divided into four groups of 10 rats each and treated for 16 weeks as follows: baseline control group fed normal food (C); acidic high-protein diet supplemented group (chronic acidosis, CA group), bicarbonate-based alkaline formula (Basenpulver, Named, Italy) supplemented chronic acidosis (BB-CA) and citrate-based alkaline supplement (CB-CA). Throughout the supplementation period, rats were put on a treadmill training mimicking a moderate level of exercise. In the CA group, 24-hour urinary calcium (Ca) and phosphorus (P) excretion were increased over 30 percent (p<0.05 vs normal diet controls). However serum Ca was not significantly changed. Femural and tibial BMD and BMC was significantly decreased in the CA group (p<0.05) but both alkaline supplementations prevented such phenomenon (p<0.05 vs CA), without significant difference between the two formulations although the BB-CA group showed significantly more preserved trabecular bone volume (p<0.05 vs CB-CA group). An increased level of over 50 percent of urinary Dpd observed in the CA group (p<0.001) was reverted to normal by both supplementations (p<0.001 vs CA group). The same applied to urinary net acid excretion (p<001) with BB-supplementation performing better than CB-supplementation (p<0.05). Moreover, while the latter did not modify Nterminal telopeptide value, BB-supplementation significantly normalized this parameter (p<0.05 vs CA group) which exercise and acidic protein diet had modified (p<0.01 vs control diet). Overall, the present study shows that a bicarbonate-based alkaline formula, when administered to a dose amenable to clinical use, may significantly protect bone structure in exercising aged animals to a greater extent than a quali/quantitavely similar citrate-based formula.

Beneficial nutraceutical modulation of cerebral erythropoietin expression and oxidative stress: an experimental study.

The main object of this study is to examine the effect of Klamin®, a nutraceutical containing phenylethylamine, phycocyanins, mycosporine-like aminoacids and aphanizomenon flos aquae-phytochrome on the learning and memory ability, the oxidative status and cerebral erythropoietin and its receptor EPO/EPOR system in prematurely senescent (PS) mice. A total of 28 PS mice, selected according to a prior T-maze test, and 26 non-prematurely senescent mice (NPS) mice were chosen. PS animals were divided into 3 groups and followed for 4 weeks: A) normal chow diet; B) added with Klamin® at 20 mg/kg/day (low dose); C) added with Klamin® at 100mg/kg/day (high dose). A further group of NPS mice given either normal food (group D) or high dose Klamin® (group E) was also considered. The behavioral procedures of spatial learning ability (Morris test) showed that PS mice had significantly longer learning time as compared to their NPS counterpart (p<0.01), but this effect was prevented especially in mice supplemented with high-dose Klamin® (p<0.05) which improved performances in NPS mice (p<0.05). High-dose Klamin® supplementation restored the depleted total thiol concentration in the brain observed in PS mice while normalizing their increased malonildialdehyde level (p<0.05). Moreover, the high-dosage only caused a significant upregulation of EPO/EPOR system both in PS and in NPS animals (p<0.05). Taken together, these data suggest that this specific alga Klamath extract has considerable antioxidant and adaptogenic properties, also through a stimulatory effect of cerebral EPO/EPO system.

Redox balance signalling in occupational stress: modification by nutraceutical intervention.

There is increasing evidence that psychosocial stress can be viewed as a system-wide derangement of cellular homeostasis, with heightened oxidative stress and triggered proinflammatory mechanisms. The aim of this study is twofold: a) to replicate findings that psychological stress increases oxidative damage and b) to determine whether a fermented papaya preparation known to exert significant protective antioxidant properties could buffer such increases in nuclear DNA damage while also inducing epigenetic protective mechanisms. Twenty-eight sedentary men and women (age range: 28-52), who reported living a stressful lifestyle but with an overall positive attitude, were recruited for this study. Chronic diseases as well as severe burnout and use of drugs for anxiety constituted exclusion criteria. Subjects were supplemented for 1 month with 9 g/day (4.5 g twice a day) of a certified fermented papaya preparation. All subjects were given a stress and sleep quality questionnaire together with a diet and life style assessment. Blood was collected at 2 and 4 week, erythrocyte and leukocyte were separated to assess redox balance and heme oxygenase-1 (HO-1) gene expression while bilirubin oxidized metabolites (BOMs) were tested in the urine. Stressed individuals showed a significant abnormality of redox status with increased MDA of erythrocyte and increased level of 8-0HdG in leukocyte and BOMs excretion (p<0.05). Nutraceutical supplementation brought about a normalization of such values already at the 2 week observation (p<0.05) together with a significant upregulation of HO-1 (p<0.01). Taken together, the results of this study confirm that stressful occupational life per se, without any overt psychiatric illness, may be associated to increased oxidative stress. Supplementation with functional food affecting redox regulation may be part of the therapeutic armamentarium to be considered in this clinical setting.

Effect of a fermented nutraceutical on thioredoxin level and TNF-alpha signalling in cirrhotic patients.

The aim of this study is to gain further insights into the possible nutraceutical effect on redox balance via thioredoxin (Trx) modulation and on the intrinsic susceptibility of monocytes to generate an inflammatory response. The study group consisted of thirty-two patients with compensated Child A-C, HCV-related cirrhosis. The patients were supplemented for 6 months with 6g/day of a certified fermented papaya preparation (FPP). Fifteen unsupplemented, age/gender-matched healthy subjects served as controls. The patients filled in a detailed diet-life style questionnaire, and blood samples were collected to test routine biochemistry, Trx, redox status (GSH, GSSG, GSH/GSSG ratio, 4-HNE and alpha-tocopherol). Moreover, isolated monocytes were tested for ex-vivo LPS-stimulated TNF-alpha production and TNF-alpha mRNA. As compared to control, patients with liver cirrhosis showed a significantly higher serum level of Trx. A significant correlation occurred with GSH/GSSG ratio in Child B and C patients. FPP supplementation brought about a significant reduction of Trx with levels comparable to the ones of healthy controls. Ten patients Child C (31.2 percent) showed borderline low levels of alpha-tocopherol while all cirrhotic patients, as a whole, showed a significantly abnormal redox balance. Supplementation with FPP did not modify alpha-tocopherol depletion but significantly improved redox balance parameters. Patients with liver cirrhosis showed a significantly upregulated TNF-alpha production in a time-dependent manner and this effect was more pronounced in more advanced stages of the disease and showed a significant correlation with alpha-tocopherol level. Supplementation with FPP significantly, although partially, downregulated TNF-alpha production from monocytes. Taken altogether, it would appear that the typical oxidative-inflammatory biochemical milieu of these patients is mirrored by a significant TNF-alpha upregulation at a monocyte level while a targeted nutraceutical might be a potentially amenable intervention to be part of validated scheduled treatments.

Disturbances of soluble N-ethylmaleimide-sensitive factor attachment proteins in hippocampal synaptosomes contribute to cognitive impairment after repetitive formaldehyde inhalation in male rats.

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein) complex, a four-helical bundle composed of syntaxin1 and synaptosome-associated protein 25 (SNAP25) on the plasma membrane and synaptobrevin/VAMP2 (vesicle-associated membrane protein 2) on the vesicle membrane, plays a key role in synaptic exocytosis and facilitates neurotransmission. Disturbances of SNARE proteins were uncovered in some neurodegenerative diseases, neuroendocrine disturbances and even after environmental interventions. In the present study, we evaluated the effects of formaldehyde (FA) inhalation (13.5+/-1.5 ppm, twice 30-min each day for two rounds of 14 consecutive days) on learning and memory in Morris water maze and thereafter explored the SNARE protein levels in hippocampal synaptosomes. The formaldehyde-treated rats showed learning and memory impairment in escape latency and probe trials, without mobility disturbances in Morris water maze. Using western blotting assays, we detected the SNARE proteins in hippocampal synaptosomes and identified decrease of both SNAP25 and VAMP2 after formaldehyde treatment without significant changes of another SNARE protein, syntaxin 1, and synaptic vesicle marker, synaptophysin. Furthermore, the neuronal morphology and number detected in Nissl stain and western blotting assay of neurofilament-150 and synaptophysin were not affected after FA treatment. These results suggested that the specific decrease of SNAP25 and VAMP2 in hippocampal synaptosomes served as a potential contributing mechanism underlying learning and memory impairments after repetitive formaldehyde inhalation treatment.

Protective effect of a poly-phytocompound on early stage nephropathy secondary to experimentally-induced diabetes.

Diabetic nephropathy (DN) is a severe and life-threatening complication of long-standing diabetes. As one of the main causes of end-stage renal disease, the prevention and treatment of DN in early stage, and the slowing down of DN progression are of utmost importance and are topics of several ongoing research studies. Nutraceuticals endowed with antioxidant-anti-inflammatory properties may offer an opportunity of integrative treatment for this condition. Male Wistar rats were randomly assigned to two groups. One group of rats (diabetic group) received a single tail-vein injection of STZ compound (50 mg/kg) under light anaesthesia. A protective dose of 0.5 ml of 5 percent dextrose was given intraperitoneally 30 min before the administration of STZ. One diabetic group was fed a normal pellet diet (group A) while group B was fed the diet added with DTS (panax pseudoginseng, eucommia ulmoides), (Kyotsu Jigyo, Tokyo, Japan) in the proportion of 50/25 (percent weight/weight), at the dose of 50 mg/kg/day throughout the experimental period. At the end of 8 weeks, 24-hour urine was collected for the measurement of the albumin concentration: blood samples were collected for serum biochemistry and the rats were sacrificed for kidney measurement of oxidative stress and histomorphological features. Nephrin and Macrophage Chemoattractant Protein-1 (MCP-1) gene expression were also assessed by fluorescence real-time quantitative PCR after RNA extraction and cDNA synthesis. STZ-treated animals showed significantly increased in lipid peroxidation in the kidney and in proteinuria. DTS supplementation did not affect plasma glucose but significantly decreased malonyldialdehyde (MDA) plasma level and the overall redox parameters together with a partial mitigation of proteinuria. Histological analysis showed also that DTS significantly reduced the glomerular volume together with glomerulosclerosis and interstitial fibrosis score (p less than 0.05), the latter two being correlated to proteinuria (p less than 0.05). DTS supplementation also enabled a reduction of diabetes-induced decrease of nephrin mRNA expression and a 67 percent reduction of MCP-1 mRNA up-regulation (p less than 0.01). Taken altogether, these data show that, besides the mandatory control of glycemia, intervention with a nutraceutical with antioxidant and anti-inflammatory properties may have beneficial effects when integrated in the mainstream of the therapeutic regimen.

Effect of modified alkaline supplementation on bone metabolic turnover in rats.

This study aims to determine the effects of a high protein diet and alkaline supplementation on bone metabolic turnover in rats. Eight-week-old male Sprague-Dawley rats were investigated by bone status, including bone mineral density (BMD) and biomechanical markers from blood and urine. Thirty rats were randomly divided into three groups and treated for 8 weeks as follows: baseline control group (n. 10, C), high-protein supplemented diet group (n. 10, chronic acidosis, CA group) and supplemented chronic acidosis (n.10, SCA). Diet-treated rats were fed an acidic high-protein diet and the supplementation consisted in a modified alkaline formula (Basenpulver, NaMed, Italy). At the end of the experimental period, the rats were sacrificed, blood samples were drawn and femur and tibia were removed for analysis of bone mineral density (BMD) by dual energy X-ray absorptiometry (DEXA). In the CA group, 24-hour urinary calcium (Ca) and phosphorus (P) excretion were increased 2.1-fold (p<0.05 vs normal diet controls) as well as kidney weight. However, serum Ca and P concentration, as well as urinary Dpd excretion were not significantly changed. Femural and tibial BMD was significantly decreased in the CA group (p<0.05), but alkaline supplementation prevented such phenomenon (p<0.05 vs CA). These results suggest that blood Ca and P concentrations in chronic acidosis condition during the 12-week supplementation might be maintained by hypercalciuria and hyperphosphaturia at the expenses of bone structure. However, modified alkaline supplementation is able to prevent such derangements.

Anti-inflammatory and neuroprotective effect of a phytoestrogen compound on rat microglia.

Ovariectomized Wistar rats received orally 15 mg/kg of a phytoestrogen compound (genistein, daidzein, glycitein, black cohosh, angelica sin., licorice, vitex agnus) for 2 weeks to test its ability to modulate inflammatory microglia response. Microglial proliferation was tested by trypan blue and by absorbance. Serial supernatant sampling was performed for 24 h to check TNF-alpha, IL-beta, IL-6, and TGF-beta. LPS caused a time course increase of all cytokines, with IL-beta and TNF-alpha peaking at the 12th hour, whereas IL-6 and TGF-beta peaked at the 24 h observation. Rats fed with the phytoestrogen displayed a significantly lower level of proinflammatory cytokines and a higher level of TGF-beta, as shown also by Western blot analysis. This finding may offer promise in the field of nutraceutical intervention.

Dominantly inherited beta thalassaemia intermedia caused by a new single nucleotide deletion in exon 2 of the beta globin gene: Hb morgantown (beta91 CTG>CG).

Family members in multiple generations of an Irish-American family were investigated for moderate to severe microcytic anaemia, inherited in an autosomal dominant fashion. A novel frameshift mutation of the beta globin gene was discovered. This study highlights the importance of considering dominantly inherited beta thalassemia in the investigation of anaemia, even in patients with ethnic backgrounds not usually associated with beta thalassaemia.

Outcome of ICG-assisted ILM peel in macular hole surgery.

PURPOSE: To investigate whether indocyanine green (ICG) staining of the internal limiting membrane (ILM) improves surgical outcome. METHODS: A total of 34 patients were identified who underwent macular hole surgery with ILM peeling augmented with ICG. These were matched retrospectively with 34 patients who underwent macular hole surgery without the use of ICG. Closure rates from primary and secondary procedures were compared, as were changes in Snellen and Logmar visual acuity. RESULTS: There was no significant difference between the two groups with reference to demographic features of age, sex, staging of the macular holes, and the proportion subsequently undergoing cataract surgery. The mean follow-up period was 7.7 months in the ICG group and 6.3 months in the non-ICG group. Closure rates from primary surgery were 91.2% in the ICG group and 73.5% in the non-ICG group (P = 0.056), whereas overall closure rates following further surgery were similar in both groups (94.1 and 91.2%, respectively). Logmar visual improvement above baseline was achieved in 53% of the non-ICG group compared to 82% of the ICG group (P = 0.01). Where primary hole closure occurred, there was no statistically significant difference in visual outcome between the two groups. CONCLUSION: ICG-assisted ILM peel in macular hole surgery is associated with a higher closure rate following a single surgical procedure. This difference was found to approach statistical significance (P = 0.056). Overall visual improvement was greater in the ICG group (P = 0.01); however, this reflected the higher closure rates. Visual outcomes between the two groups were comparable where primary hole closure occurred.