An increasing body of evidence implicates both brain inflammation and oxidative stress in the pathogenesis of Alzheimer's disease (AD). The relevance of their interaction in vivo, however, is unknown. Previously, we have shown that separate pharmacological targeting of these two components results in amelioration of the amyloidogenic phenotype of a transgenic mouse model of AD-like brain amyloidosis (Tg2576).In the present study, we investigated the therapeutic effects of a combination of an anti-inflammatory agent, indomethacin, and a natural anti-oxidant, vitamin E, in the Tg2576 mice. For this reason, animals were treated continuously from 8 (prior to Aβ deposition) through 15 (when Aβ deposits are abundant) months of age.At the end of the study, these therapeutic interventions suppressed brain inflammatory and oxidative stress responses in the mice. This effect was accompanied by significant reductions of soluble and insoluble Aβ1-40 and Aβ1-42 in neocortex and hippocampus, wherein the burden of Aβ deposits also was significantly decreased.The results of the present study support the concept that brain oxidative stress and inflammation coexist in this animal model of AD-like brain amyloidosis, but they represent two distinct therapeutic targets in the disease pathogenesis. We propose that a combination of anti-inflammatory and anti-oxidant drugs may be a useful strategy for treating AD. Alzheimer's disease (AD) is the most common, complex and challenging form of neurodegenerative disease associated with dementia in the elderly. Neuropathological examination of the AD brain shows extensive neuronal loss, accumulation of fibrillar proteins as extra-cellular amyloid β (Aβ) plaques, and as neurofibrillary tangles (NFTs) inside neurons . HoweverIn the present study, we examined whether the simultaneous administration of an anti-oxidant, vitamin E, with an anti-inflammatory drug, indomethacin, would exert an additive anti-amyloidogenic effect in the Tg2576 mouse model of AD-like Aβ brain amyloidosis, one of the most extensively studied mouse models of AD . Signifiin vivo and significantly reduced brain inflammation [The genotype and phenotype features of the heterozygote Tg2576 mice that we studied here have been described in earlier reports on these mice from our group . Mice weammation . The higammation . During Animals were anesthetized and euthanized following procedures recommended by the Panel on Euthanasia of the American Veterinary Medical Association. They were always perfused intra-cardially for 30 min with ice-cold 0.9% phosphate buffer saline (PBS), containing EDTA (2 mM/L) and BHT (2 mM/L), pH7.4. Brains were removed and one hemisphere was fixed by immersion in 4% paraformaldehyde in 0.1 M PBS (pH7.4) at 4°C overnight, blocked in the coronal plane, and embedded in paraffin as previously described for immunohistochemistry ,17, The 2α-VI by ion chemical ionization gas chromatography/mass spectrometry assay, as previously described [2 and TxB2 by a standardized ELISA kit following the manufacturer's instructions (Cayman Chem. Com.). Briefly, extracts were diluted with acetate buffer and purified through an affinity column. The purified samples were evaporated, re-dissolved in the assay buffer and applied to 96-well plates pre-coated with goat anti-serum IgG and incubated with PGE2 or TxB2 monoclonal antibodies. The plates were rinsed with washing buffer and developed using Ellman's reagent for 60–90 min at room temperature with gentle shaking. Specific concentrations were determined spectrophotometrically and expressed as pg/mg tissue.Tissue samples were minced and homogenized, and total lipid extracted with ice-cold Folch solution . Lipids were subjected to base hydrolysis by adding aqueous 15% KOH and then incubated at 45°C for 1 hr for measurement of total iPFescribed ,16,17. IIL-1β levels were measured by a standardized sandwich ELISA kit following the manufacturer's instructions (Endogen Pierce). Briefly, equal amounts of sample were loaded onto 96-well plate pre-coated with monoclonal antibody against mouse IL-1β overnight at 4°C. The plates were rinsed three times with washing buffer and developed with streptavidin-horseradish peroxidase (HRP) . SpecifiTotal protein carbonyls in tissue were determined by using the Zenith PC test kit according to the manufacturer's instructions (Zenith Tech.) . BrieflyAn aliquot of brain homogenates was electrophoresed on a 10% acrylamide gel under reducing conditions. Protein were transferred to a polyvinylidene membrane before blocking in 10% nonfat dry milk for 2 hr. Blots were incubated with monoclonal antiboby against glial fibrillary acidic protein (GFAP) (2.2B10) (1:1,000), or an anti-beta actin (1:5,000) antibody overnight at 4°C. After three rinses, blots were incubated with HRP-conjugated goat anti-mouse for 45 min before development with chemiluminescent detection system using ECL (Amersham). Bands were quantified using densitometric software . The anti-GFAP is a monoclonal antibody, and its characterization has previously been published  . Subsequently, sections were incubated with a biotinylated antibody against Aβ (4G8) (1:10,000 dilution), at 4°C overnight [Serial 6-μm-thick paraffin sections were cut throughout each brain, and mounted on APES-coated slides. Sections were deparaffmized, hydrated, rinsed with PBS and pre-treated with formic acid (88%) for 10 min to antigen retrieval, and with 3% Hvernight ,17. Sectvernight ,16,17. At test corrected for multiple comparisons. Significance was set at p < 0.05.Data are expressed as mean ± standard error of mean (S.E.M.), analyzed by analysis of variance (ANOVA), and subsequently by student unpaired 2-tailed 2 and suppression of TxB2 levels in tissue homogenates from total cortex and hippocampus (Table 2α-VI (a reliable biomarker of lipid peroxidation), as well as protein carbonyls (known biomarkers of protein oxidation) were both significantly decreased  added to their diet, plus indomethacin (l0 mg/liter) in their water, and they were treated until they were 15 months old. Notably, at 8 months of age, the Tg2576 mice show elevated brain levels of soluble and insoluble Aβ as well as isoprostanes, relative to their non-transgenic littermates, but they show no evidence of any brain Aβ deposits, while following the initial onset of mature plaque-like brain deposits at about 11–12 months of age, the Tg2576 mice show abundant Aβ deposits and higher levels of isoprostanes in neocortex and hippocampus a 15 months of age ,15,20. AWestern blot analysis was used to determine the effect of the drug treatment on GFAP levels, a marker of astrocytosis . These lNext, we assessed the effect of indomethacin and vitamin E on brain levels of soluble and insoluble Aβ1-40 and Aβ1-42. As expected for their age, Tg2576 mice on placebo showed elevated levels of both forms of these peptides in their cortex and hippocampus Figure , whereas2-isoprostanes (lipid peroxidation); protein carbonyls, nitrotyrosine (protein oxdidation); 8-hydroxy-2'-deoxyguanosine (DNA oxidation) [Caernorhabditis elegans model, which over-expresses Aβ1-42 [There is substantial evidence implicating both oxidative stress and inflammatory mechanisms in AD pathogenesis. Evidence for oxidative stress derives from both human (post-mortem and living patients) studies, and transgenic mouse models of the disease. There is a long list of surrogate markers of reactive oxygen species-mediated injury that have been found increased in the brain and cerebrospinal fluid of AD patients. It includes, just to mention a few, malondialdehyde, 4-hydroxynonenal, Fidation) -5. Transidation) ,21. Oxids Aβ1-42 ,22,23. Fs Aβ1-42 ,25. TakeInflammatory mechanisms are also operative in the AD brain and significantly contribute to the pathophysiology of the disease. Although classical defined inflammation, including such features as edema and neutrophil invasion, is not seen in the AD brain, hallmark of innate immune response are constant elements of the neuropathology associated with brain degeneration in AD . FurtherLim et al. first reported that chronic administration of the NSAID ibuprofen to Tg2576 reduces total Aβ levels, amyloid burden and brain inflammation . More reIn the present study, we extended these previous observations by examining whether administration of indomethacin in combination with vitamin E would result in a better anti-amyloidotic effect. Our findings show that soluble Aβ1-40 and Aβ1-42 levels were reduced by ~65%, while the insoluble fractions were decreased by ~55%. Consistently, we observed a better and more diffuse effect also on the amount of amyloid deposited in the brain at the end of the study. Finally, the two drugs together produced an additive affect on brain inflammation and oxidative stress ,17.Our results confirm previous observation where low-dose curcumin, a drug with reported both anti-oxidant and anti-inflammatory activities, reduced total Aβ and plaque burden . HoweverIn our study, we used two different drugs with a more restricted therapeutic target to provide further evidence that both oxidative stress and inflammation are indeed functionally relevant in the development of the phenotype of these animals. However, we also provide new information on the critical issue of the in vivo relationship between these two events. Thus, our results suggest that brain inflammation and oxidative stress are two separate events, which work in concert to modulate the development of this AD-like brain Aβ amyloidosis model. Previously, we have shown that a full dose of indomethacin alone despite a significant reduction in brain inflammation had only a marginal effect on brain oxidative stress in the Tg2576 mice . This fiIn summary, our findings support the hypothesis that oxidative stress and inflammation represent important but distinct therapeutic targets in AD-like amyloidosis. We conclude that a combination of therapeutic agents targeting these different disease-modulating mechanisms might be rationally evaluated in the prevention or therapy of AD in humans.AD: Alzheimer's diseaseAβ: Amyloid β peptideTg: Transgenic mouse modelNSAIDs: Non-steroidal anti-inflammatory drugs2: Prostaglandin E2PGE2: Thromboxane A2TxBGFAP: Glial fibrillary acidic proteinIL-1β: Interleukin 1-β2α-VI: Isoprostane F2α-VIIPFThe authors declare that they have no competing interests.Yuemang Yao and Cinzia Chinnici have made substantial contribution to the acquisition of data and biochemical analyses. Hanguan Tang contributed to the immunohistochemical analyses. John Q. Trojanowski and Virginia M-Y Lee have been involved in the interpretation of data, and the critical revision of the manuscript for intellectual content. Domenico Praticò has been involved in the conception and design of the studies, interpretation of data, drafting and critical revision of the manuscript.


In this study, we derive an expected value for f, starting from a nearly neutral model of weak selection, and then assess the biological interpretation of this factor with evolutionary simulations.The f = 0.5  is derived for sequences that are evolving under the nearly neutral model of this study. However, this expectation is sensitive to violations of its underlying assumptions as illustrated with the evolutionary simulations.An expected value of f. However, as f is affected by a number of factors that limit its biological interpretation, this factor should normally be estimated as a free parameter rather than fixed a priori in a +gwF analysis.This study illustrates how selection, drift, and mutation at the population level can be linked to the rate matrices of models for sequence evolution to derive an expected value of  Goldmaet al.  describeet al. , and K2Pet al. ). At theqij refers to the substitution rate from nucleotide i to j, πi and πj correspond to their equilibrium base frequencies, and sij is the exchangeability between the two. In the +gwF variants, the substitution rate becomes more dependent on the ending nucleotide as f decreases from 1 to 0, with f = 0 for the classic F81-type models.where f parameter and evolutionary rate matrices of the +gwF variants. These theoretical derivations lead to an expected value of f = 0.5. However, as illustrated with simulations, the f parameter is complex and thus its biological interpretation must be considered with caution.This study starts with a population genetics model to derive equations that link weak selection, genetic drift, and mutation to the N, a neutral mutation has a probability of 1/2N of becoming fixed in the population. However, because of drift, even slightly deleterious mutations can become fixed, but at a probability of less than 1/2N. Advantageous mutations have higher fixation probabilities than neutral mutations. In the nearly neutral model, the distribution of alleles is determined by an equilibrium of selection, drift, and mutation.The nearly neutral model of molecular evolution states that most DNA mutations of longer-term evolutionary consequence are under weak selection and are therefore prone to drift ,8. For aDrosophila [sA, sC, sG, and sT. The differences between these selection coefficients will be very close to zero, since no strong selection is expected.Consider a number of sites under identical evolutionary constraints and with a bias in nucleotide distribution. Assume that weak selection and drift are the causes of this bias; e.g., as for the codon usage biases in micro-organisms and osophila ,10. In oosophila ,12. Let i to j, with a selective advantage of s = sj - si (a selective disadvantage exists when s is negative). For a population of size N and an effective size of Ne, Kimura [Consider a mutation from nucleotide , Kimura  showed ts ≠ 0. For s = 0, we have P(s) = 1/2N. This approximation is valid for small values of s, which is the case here.when i to j is proportional to P(sj - si):The substitution rate from nucleotide qij = 2N μijP(sj - si),     (3)μij is the mutation rate from i to j. For different i and j, μij can vary because of unequal transition versus transversion rates (for example). Furthermore, let us assume that the mutation rate is the same for either direction of substitutions between i and j. This assumption is necessary to maintain the widely used condition of time reversibility in the evolutionary process, which thereby keeps the following derivations tractable [where ractable ,13.We then have:qij/qji can be written as a function evaluated at sj divided by the same function evaluated at si, evolution is time reversible according to this model with:Since c and c' are constants with c' = -l/4Ne log c, which will be chosen to make the equilibrium frequencies sum to one. The substitution rates can now be approximated as:Here, sij = μij, this equation reduces to equation (1) with f = 0.5 and an adjustment factor of:Given an exchangeability of π, with a horizontal tangent around πj/πi = 1 and a slight bending downwards when deviating from this value . Simulations consisted of four sequences of length 10,000 and relied on a symmetric rooted phylogeny with all branch lengths equal to 0.10 expected substitutions per site under the model in question [i.e., ((seq1:0.10, seq2:0.10):0.10, (seq3:0.10, seq4:0.10):0.10)]. Violations of the weak selection model were incorporated in the simulations by: (1) heterogeneous sequences with sites drawn from different equilibrium base frequencies; (2) populations in disequilibrium due to changing Ne; and (3) an accelerated C to T substitution rate. Estimates of f for the simulated sequences were made with the K2P+gwF model. Forty simulations were run for each test condition, with the results for the f estimates summarized as their means and twice their standard errors. In the first set of simulations, six categories of sites with different equilibrium distributions were considered . In contrast, for the simulated heterogeneous sequences , their values of f varied significantly in either direction from 0.5. Analogous results were obtained for the simulations of homogeneous and heterogeneous sequences under the HKY model (with f = 0.0 instead of 0.5). Thus, the value of f can vary considerably when heterogeneous sequences are analyzed with a +gwF model. Here, such deviations are a consequence of using a single rate matrix to analyze sequences that were derived from two or three different ones.Evolutionary simulations were conducted to examine the effects of violating certain assumptions in the above model of weak selection. Unless otherwise noted, these simulations were based on the K2P+gwF model with Ne was kept constant until the time of the most recent common ancestor for the four simulated sequences. Then, Ne was either left unchanged or was suddenly changed by a certain factor. The latter was done by replacing the rate matrix derived from equation (4), resulting in new equilibrium frequencies of the nucleotides. When Newas kept constant, the selective pressures and drift were left unchanged, thereby maintaining the same starting equilibrium frequencies throughout the phylogeny. Thus, the corresponding f estimates did not significantly differ from 0.5 (Fig. Ne lowered the value of f as the efficiency of selection was increased relative to drift [In the second set of simulations, 0.5 Fig. . In contequation , resultif in either direction from 0.5, even though their sequences were simulated in equilibrium (Fig. f can vary considerably when the rates for reciprocal mutations are unequal.In the third set of simulations, an acceleration in the C to T substitution rate was incorporated, thereby modeling an increase in their mutation rate due to the deamination of methylated C's in CpG pairs . The intium Fig. . Thus, tf parameter and rate matrices of the +gwF variants for sequence evolution. Our weak selection model relies on the fixation probabilities of mutant alleles with additive genie selection and equal mutation rates for reciprocal substitutions. What is now needed are additional studies that link other population genetics models to the +gwF variants [f parameter and +gwF rate matrices.This study illustrates how selection, drift, and mutation within a population can be linked to the variants . For exavariants , which ff parameter is complex and can be influenced by a number of different factors [f ill need to be estimated as a free parameter rather than fixed beforehand.Collectively, the three sets of simulations highlight that the  factors . This coGoldman and Whelan  focused Both authors contributed to the conception and design of this study and to the writing, reviewing, and final approval of this article. B.K. performed the simulations and parameter estimations.


The complement system forms the central core of innate immunity but also mediates a variety of inflammatory responses. Anaphylatoxin C3a, which is generated as a byproduct of complement activation, has long been known to activate mast cells, basophils and eosinophils and to cause smooth muscle contraction. However, the role of C3a in the pathogenesis of allergic asthma remains unclear. In this review, we examine the role of C3a in promoting asthma. Following allergen challenge, C3a is generated in the lung of subjects with asthma but not healthy subjects. Furthermore, deficiency in C3a generation or in G protein coupled receptor for C3a abrogates allergen-induced  responses in murine models of pulmonary inflammation and airway hyperresponsiveness. In addition, inhibition of complement activation or administration of small molecule inhibitors of C3a receptor after sensitization but before allergen challenge inhibits airway responses. At a cellular level, C3a stimulates robust mast cell degranulation that is greatly enhanced following cell-cell contact with airway smooth muscle (ASM) cells. Therefore, C3a likely plays an important role in asthma primarily by regulating mast cell-ASM cell interaction. H2 cytokine producing T cells, mast cells and ASM cells play central roles in the pathogenesis of asthma [Asthma, a complex airway inflammatory disease, is characterized by bronchoconstriction, airway hyperresponsiveness (AHR) and remodeling. Current consensus suggests that Tf asthma -7. The cf asthma ,9. A rolf asthma .C3a levels are elevated in bronchoalveolar lavage (BAL) fluid after segmental allergen challenge in asthmatic but not healthy subjects -14. FurtH2 cytokine production and eosinophil recruitment in BAL when compared to wild-type mice. Additionally, guinea pigs with a natural defect in C3aR expression were also protected from AHR in response ovalbumin to challenge with no effect on airway inflammation [Studies with animal models provided compelling evidence for C3aR activation in the development of AHR and inflammation. Humbles et al., , showed ammation . These iammation ,17,18.Aspergillus fumigatous and challenged intranasally with allergen, these mice experienced substantial decreases in both AHR and airway eosinophilia relative to wild-type mice. Furthermore, BAL levels of TH2 cytokines (IL-4, IL-5, IL-13), IgE titres and mucous production were all significantly reduced in C3aR (-/-) mice. Allergen-challenged C3 (-/-) mice also display diminished AHR, lung eosinophilia and TH2 cytokine production when compared to wild-type mice [Recent studies using C3aR (-/-) mice provided new insights on the role of C3a on both AHR and airway inflammation . When seype mice . These fAspergillus fumigatous and mast cell tryptase also activate the complement pathway directly [Increased level of C3a in BAL of subjects with asthma implies a potential role for this apaphylatoxin in promoting airway inflammation. However, the cells responsible for C3a generation and the airway effector cells stimulated by C3a remain unknown. Plausibly, antibody generated during antigen sensitization may interact with allergen to activate the classical complement pathway. Additionally, airway epithelial cells and pulmonary macrophages secrete both C3 and several components of the alternate pathway of complement (factors B, H, and I and properdin) -23. Thusdirectly ,24-26. TH2 cytokine response to intraperitoneal ovalbumin sensitization. Furthermore, C3a had no effect on TH2 cytokine production in response to T cell receptor ligation. Further, Taube et al., [H2 cytokine production and lung inflammation. Additionally, a small molecule antagonist of C3a receptor, when administered after sensitization but before challenge also caused significant inhibition of airway inflammation [Both antigen-presenting cells (APCs) and activated T cells express C3aR -30, rais et al.,  showed tammation . These f+-derived primary mast cell cultures [4 generation (Thangam, B and Ali, H, unpublished data). These findings suggest that allergen induces mast cell degranulation by at least two mechanisms: cross-linking of FcεRI and via C3a generation following complement activation by allergen protease , which induce mast cell chemotaxis, survival and differentiation ,48. Inteytokines -52. ThusC3a has long been recognized as an agent that evokes force generation in smooth muscle. In guinea pigs, C3a-induced contraction of lung parenchyma may involve indirect effects of histamine and arachidonic acid metabolites . In miceRecently, investigations showed that human mast cells but not human or murine ASM express C3aR . InteresH2 lymphocyte and their cytokines IL-4, IL-5 and IL-13. These cytokines play key roles in the recruitment and activation of eosinophil, mucous production and IgE synthesis. Allergen challenge of sensitized C3 (-/-) and C3aR (-/-) mice decreased production of TH2 cytokines in BAL and substantially reduced recruitment of T cells, eosinophils and neutrophils in lung tissue [H2 effector function in murine model of allergen-induced inflammation. Accordingly, in human mast cells, C3a stimulates the production of MCP-1, RANTES [H2 cytokine and chemokine production in asthma  declare that they have no competing interests.


Capsule related complications are common following pediatric cataract surgery. We report a new technique of multiple anterior capsulorhexisotomies after lens aspiration and intraocular lens (IOL) implantation.After performing automated lens aspiration, an IOL was implanted into the capsular bag. A bent 26 gauge needle was introduced through one side port and multiple small cuts were made in one half of the circumference of the anterior capsular rim by making a radial movement of the needle tip centripetally over the margin of the anterior capsular rim. The needle was again introduced through the other side port and multiple similar cuts were made in the other half thereby creating nearly 20 – 30 cuts at the margin of the anterior capsular rim.The mean size of the primary capsulorhexis was 4.33 ± 0.20 mm. A uniform enlargement of the capsulorhexis could be performed in all the eyes without peripheral extension in any of the eyes. There was no damage to the posterior capsule and no scratch mark on the IOL. In one eye, the primary capsulorhexis was slightly eccentric, though it was covering the IOL optic all around. The rhexisotomies in this eye were limited to the capsular rim that was overlapping more on the IOL optic .The technique of postage stamp anterior capsulorhexisotomies is a feasible technique in pediatric cataracts. Pediatric cataract surgery has always been a challenge for the anterior segment surgeons and short term and long term complications have been reported in the literature . AutomatHowever, most prefer using Utrata capsulorhexis forceps to complete the capsulorhexis after an initial nick in the anterior capsule with a bent 26 gauge needle.We herein, describe a new technique of postage stamp multiple capsulorhexisotomies, a modification in the primary capsulorhexis after performing lens aspiration with intraocular lens implantation in pediatric cataracts.We performed this technique of anterior capsulorhexisotomies in 6 eyes of 4 children; 2 patients with bilateral developmental cataract and 2 with unilateral post-traumatic cataract.Surgery was performed in all the eyes under general anesthesia. A clear corneal 3.2 mm 3-plane tunnel was created superiorly. Two side ports were created at 10 and 2 o'clock positions using a microvitreoretinal blade (Alcon laboratories, Fort Worth, TX). Trypan blue (Vision blue, DORC, Netherlands) was injected (0.1 ml of 0.1%) into the anterior chamber under air bubble to stain the anterior capsule and was completely washed out after 15 seconds by balanced saline solution. Anterior capsulorhexis was initiated with a bent 26 gauge needle and completed by Utrata capsulorhexis forceps to create a circular, 4.0 – 4.5 mm, central capsulorhexis. The size of the capsulorhexis was measured on a television monitor according to a previously described method [Sodium hyaluronate 1.4% was injected under the anterior capsular rim. A bent 26 gauge needle was introduced through one side port and multiple small cuts were made in one half of the circumference of the anterior capsular rim by making a radial movement of the needle tip centripetally over the margin of the anterior capsular rim taking care not to put any scratch mark on the IOL. The needle was again introduced through the other side port and multiple similar cuts were made in the other half thereby creating nearly 20 – 30 cuts all around circumferentially at the margin of the anterior capsular rim  years and all patients were males. The mean size of the primary capsulorhexis was 4.33 ± 0.20 mm. The nicks at the margin of the anterior capsular rim could be performed successfully with a bent 26 gauge needle in all the eyes. A uniform enlargement of the capsulorhexis could be performed in all the eyes without peripheral extension in any of the eyes. No eye suffered damage to the posterior capsule. There was no scratch mark of the needle on the optic of any IOL. In one eye, the primary capsulorhexis was slightly eccentric, though it was covering the IOL optic all around. The rhexisotomies in this eye were limited to the capsular rim that was overlapping more on the IOL optic .Size of anterior capsulorhexis has always been a matter of debate for automated lens aspiration in pediatric cataracts. If the anterior capsulorhexis opening is small, there is risk of anterior capsular opacification, capsular contraction syndrome and phimosis of the anterior capsular opening, decentration of the IOL and capsular bag hyperdistension ,5. DespiWe have observed that after curvilinear capsulorhexis, capsular opacification and fibrosis is most marked at the margin of the anterior capsular rim. We performed capsulorhexisotomies to prevent phimosis of the anterior capsular opening and capsular contraction syndrome, as these small nicks act as relaxing incisions. Since there were multiple nicks all around, the direction of the force is well distributed. The vector of the force generated at the opacified margin of curvilinear capsulorhexis is directed inwards, while after performing multiple nicks in this margin (capsulorhexisotomies), the vector is directed in both inward as well as outward directions. Therefore, there is less chance of development of capsular contraction syndrome. More over, there is negligible chance of adhesion between the anterior and posterior capsules as the anterior capsular rim rests well on the IOL optic.The postage stamp multiple anterior capsulorhexisotomies is a feasible and safe technique after lens aspiration and IOL implantation in pediatric cataracts.The author(s) declare that they have no competing interests.JST designed the study and performed surgeries. RS performed the data collection and wrote the manuscript. NS followed up the patients. RBV performed the surgeries.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Signal transducers and activators of transcription (STATs) are involved in growth regulation of cells. They are usually activated by phosphorylation at specific tyrosine residues. In neoplastic cells, constitutive activation of STATs accompanies growth dysregulation and resistance to apoptosis through changes in gene expression, such as enhanced anti-apoptotic gene expression or reduced pro-apoptotic gene expression. Activated STAT3 is thought to play an important role in prostate cancer (PCA) progression. Because we are interested in how persistently-activated STAT3 changes the cellular phenotype to a malignant one in prostate cancer, we used expression vectors containing a gene for constitutively-activated STAT3, called S3c, into NRP-152 rat and BPH-1 human benign prostatic epithelial cells.We observed that prostatic cell lines stably expressing S3c required STAT3 expression for survival, because they became sensitive to antisense oligonucleotide for STAT3. However, S3c-transfected cells were not sensitive to the effects of JAK inhibitors, meaning that STAT3 was constitutively-activated in these transfected cell lines. NRP-152 prostatic epithelial cells lost the requirement for exogenous growth factors. Furthermore, we observed that NRP-152 expressing S3c had enhanced mRNA levels of retinoic acid receptor (RAR)-α, reduced mRNA levels of RAR-β and -γ, while BPH-1 cells transfected with S3c became insensitive to the effects of androgen, and also to the effects of a testosterone antagonist. Both S3c-transfected cell lines grew in soft agar after stable transfection with S3c, however neither S3c-transfected cell line was tumorigenic in severe-combined immunodeficient mice.We conclude, based on our findings, that persistently-activated STAT3 is an important molecular marker of prostate cancer, which develops in formerly benign prostate cells and changes their phenotype to one more closely resembling transformed prostate cells. That the S3c-transfected cell lines require the continued expression of S3c demonstrates that a significant phenotypic change occurred in the cells. These conclusions are based on our data with respect to loss of growth factor requirement, loss of androgen response, gain of growth in soft agar, and changes in RAR subunit expression, all of which are consistent with a malignant phenotype in prostate cancer. However, an additional genetic change may be required for S3c-transfected prostate cells to become tumorigenic. In benign cells, the signaling by STAT3 is under tight regulation, so that the signal delivered to the cell is transient. However aberrant signaling by STAT3 has been noted in many types of malignancies, such as myeloma, head and neck cancer, breast cancer, and prostate cancer [Signal transducers and activators of gene transcription (STATs) are, as their name suggests, proteins that regulate gene expression by affecting transcription. They are part of the signal transduction pathway used by many growth factors and cytokines, and are activated by phosphorylation of tyrosine and serine residues by up-stream kinases . For exae cancer -9. Such e cancer . Importae cancer -13. Thuse cancer ,15.trans retinoic acid in human prostate cancer lines [Prostate cancer (PCA) is the second most frequently diagnosed non-cutaneous malignancy in American males, affecting approximately 35% of them according to recent data ,17. Thiser lines ,35To this end we are studying the oncogenic role of STAT3 activation in rat prostate epithelial cell lines NRP-152  and humaTwo weeks after transfection with either pIRES or pIRES-S3c and selection with G418, no surviving cells were observed in the wells that received Clonfectin only. Growth of cells was observed in all wells that received either of the plasmids plus Clonfectin. Transfected cells were expanded for further analysis in complete medium. A summary of cells and clones and what their phenotypes were is given in Table NRP-152 cells require a variety of growth factors and additives in their medium ; 152-pIBPH-1 cells grow in RPMI-1640 supplemented with bovine serum; therefore this line does not have growth factor dependence to begin with. BPH-pIRES and BPH-S3c cells, aside from exhibiting G418 resistance, expressed EGFP, but only BPH-S3c expressed the FLAG epitope of the S3c gene. The evidence for these observations given in Table 2)', as well as the enhanced green fluorescent protein fluorescence intensities of transfected cells, are shown. Panel A displays the anti-FLAG fluorescence intensity of 1 representative clone of 152-S3c (thin line) compared to untransfected NRP-152 cells (thick line); approximately 95% of the 152-S3c cells stained with the anti-FLAG antibody. Similary, Panel B shows the fluorescence intensity of anti-FLAG-stained BPH-1 cells (thin line) compared to anti-FLAG-stained BPH-S3c clone (thick line), where approximately 76% of the BPH-S3c cells stained with the anti-FLAG antibody. Panels C and D display the EGFP fluorescence for clones of 152-S3c and BPH-S3c cells, compared with untransfected cells, respectively. In Panel C, the thick line shows the fluorescence intensity of EGFP in 152-S3c and the thin line shows the lack of EGFP fluorescence in the untransfected NRP-152 cells. Approximately 67% of the 152-S3c cells showed EGFP fluorescence. In Panel D, the thin line shows the EGFP fluorescence intensity of BPH-S3c cells, while the thick line shows it for untransfected BPH-1 cells. Approximately 45% of the BPH-S3c cells showed fluorescence due to EGFP. We concluded that in addition to antibiotic resistance, the transfected cells expressed markers flanking the S3c gene, and therefore we could attribute any change in phenotype of the cells to the expression of the S3c, in comparison to the vector-transfected cells. Panel E shows the results of immunoprecipitation with anti-FLAG Ab, followed by Western blot to detect EGFP. We used anti-FLAG Ab for the immunoprecipitation because (1) a S3c-specific Ab is not available, and (2) because all cells express STAT3. Thus, because expression of FLAG equates with expression of S3c specifically, immunoprecipitating with anti-FLAG would reveal the S3c-expressing cells. As seen in Figure After no viable cells were observed following antibiotic treatment, we analyzed transfected cells for the presence of the markers flanking the S3c gene on the plasmids used, FLAG and EGFP. The analyses were done by flow cytometry on a FACScan; also by Western blot using specific Abs, and the results are presented in Figure To demonstrate that 152-S3c cells grew in the absence of growth factors required by untransfected NRP-152 cells, transfected and untransfected NRP-152 cells were grown in microtiter wells. Proliferation was quantified by the oxidation of MTT after 48 hr. Figure In order to show that the persistent expression of activated STAT3 was required for the survival of the transfected cells, as we have previously shown for hormone-refractory prostate cancer cells lines ,12, we tIn prostate cancer cell lines and archived specimens, we previously found that RAR-β and -γ have decreased mRNA levels, while RAR-α mRNA increased, relative to non-malignant prostate cell lines and the normal margins of the same specimens ,35. ThisIn many human prostate cancers, overexpression of the androgen receptor has been noted ,43. TherIn non-malignant cells, the activation of STAT3 is effected by a specific upstream kinase, JAK1 or JAK2 or sometimes Tyk2. Previously we had shown that the constitutive activation of STAT3 in NRP-154 cells rendered those cells insensitive to apoptosis induced by the JAK2 inhibitor AG490 . In ordeAs an in vitro indication of tumorigenic potential, soft agar cloning assays were performed as described . S3c-traBased on our previous data, especially the soft agar cloning data, we expected that 152-S3c cells would form tumors in SCID mice. However, in 3/3 experiments (in two them, Matrigel was used to enhance tumorigenicity of the cells), an average of 1/5 mice developed tumors; these were 1 mm in diameter or less. We chose to use only transfected NRP-152 cells for these experiments, because in certain in vivo environments, untransfected BPH-1 cells have been observed to form tumors . We concWe have demonstrated that NRP-152 and BPH-1 cells transfected with a constitutively-activated form of the STAT3 gene, S3c, gained a phenotype which more closely resembled that of NRP-154 cells. Specifically, the transfected cells expressed resistance to the antibiotic G418, and also expressed the FLAG epitope, as revealed by intracellular flow cytometry following staining with anti-FLAG Ab in Figure Parental NRP-152 cells are fastidious in their growth factor requirement, whereas NRP-154 cells and 152-S3c clones grew in medium supplemented only with serum Figure ; the redAs for RAR expression, we observed decreased mRNA levels of RAR-β and -γ, but increased RAR-α expression in S3c-transfected NRP-152 cells; the results shown in Figure In terms of androgen receptor function, S3c expression in BPH cells changed their response to androgens so that BPH-S3c cells were no longer stimulated by DHT, and the growth of BPH-S3c cells was not inhibited by flutamide treatment Table . These fc-fos, and subsequent tumorigenicity, in NIH-3T3 cells [c-fos in S3c-transfected lines can be determined. As well, Dechow and coworkers reported that transfection of S3c into mammary epithelial cells rendered those cells tumorigenic in irradiated SCID mice [Prior to performing in vivo tumorigenicity experiments, we wanted to see if S3c-transfected cells could grow in soft agar as clones. We observed that S3c expression in NRP-152 cells allowed them to grow as clones in soft agar Table . HoweverT3 cells . WhetherCID mice ; whetherOur data indicate that transfection of NRP-152 and BPH-1 prostatic epithelial cells with a gene for persistently-activated STAT3, S3c, changed the phenotype of the cells into one resembling a malignant phenotype, thereby giving even more importance to the role of activated STAT3 in the transformation of normal cells into neoplastic cells. Importantly, we found that cells expressing S3c depended on its continued expression for survival. Two kinds of evidence are presented: first, S3c-transfected cells became sensitive to the effect of antisense STAT3 oligonucleotide. When transfected with antisense STAT3, both BPH-S3c and 152-S3c underwent apoptosis. Second, the S3c-transfected cells were not sensitive to the commonly-used STAT3 inhibitors, which are really JAK inhibitors, because activation of STAT3 by the upstream JAK is not required when S3c is expressed. We observed that growth factor-dependent NRP-152 cells grew without growth factor supplementation when transfected with S3c gene, whereas the medium for vector-transfected NRP-152 cells still required supplementation with growth factors. Moreover, we observed that 152-S3c cells grew in soft agar, whereas neither vector-transfected nor untransfected NRP-152 cells did. Furthermore, we observed that the expression of RAR subunits in 152-S3c cells was different from vector-transfected and untransfected NRP-152 cells, and that the changes were consistent with what we previously observed in specimens from prostate cancer patients, as well as in primary prostatic epithelial cells compared with prostate cancer cell lines ,35. ThesNRP-152 and NRP-154 cells were the gift of Dr. David Danielpour, Ireland Cancer Center, University Hospitals, Cleveland, OH . Growth Derivation of the pBABE-S3c plasmid containing a constitutively-activated STAT3 gene, S3c  has been previously described ,45. The For transient transfections, LipoFectamine 2000 in Opti-MEM I medium (both, Invitrogen) was used according to the manufacturer's directions. For subconfluent (~50%) cells, 2 μl of LipoFectamine 2000 was used with varying amounts of antisense or sense STAT3 oligonucleotide . The oligonucleotides were left on the cells for 6 hours before cell culture medium supplemented with 30% was added to each well. Cells were incubated until assays were performed.In order to analyze clonal populations of cells, transfected cells (pIRES-S3c or pIRES-EGFP) were harvested, diluted to 10 cells/ml in complete medium, and seeded into microtiter plates at 100 μl/well. The total volume of each well was brought to 200 μl with additional medium, and the plates were incubated until growth of seeded cells was observed, usually at 10 days to 2 weeks.6 cells/100 μl permeabilization buffer) to the cells for 1 hr on ice. The cells were washed 3 times, then incubated with phycoerythrin (PE)-labeled goat anti-mouse F(ab2)'  for 1 hr on ice in permeabilization buffer. After washing 3 times, cells were resuspended in 1 ml PBS and analyzed on a Becton-Dickinson FACScan. CellQuest software was used to acquire and analyze the fluorescence. The Kolmogorov-Smirnov 2-sample test was used to determine the level of significance of the change in fluorescence intensity between control-stained (F(ab2)'-stained only) and Ab-stained populations of cells, thereby ascertaining that the populations observed in the histograms were truly separate populations of cells [Expression of the FLAG epitope engineered onto the constitutively-activated STAT3 gene in transfected NRP-152 cells was performed by intracellular flow cytometry, as described [of cells .For immunoprecipitation, cells lysed in Lysis Buffer (10 mM PBS, pH 7.4, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate (SDS), 1 mM sodium orthovanadate, 1 mM phenylmethyl-sulfonyl fluoride, 40 μg/ml aprotinin) were precleared with Protein A/G agarose (Santa Cruz Biotechnology), then precipitated with 1–5 μg rabbit Ab  plus Protein A/G (Santa Cruz Biotechnology) agarose overnight. After washing, the beads were eluted by heating in Laemmli buffer for 5 min at 95°C, followed by electrophoretic separation on 12% SDS-polyacrylamide gels (Novex Nu-PAGE pre-cast gels). Transfer of separated protein species to nylon membrane (Millipore) was followed by blocking in 10% non-fat dry milk in TBST (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 0.3% Tween 20). Incubation of the membrane with rabbit Ab was followed by incubation with alkaline phosphatase-linked goat anti-rabbit antibody (Amersham ECF kit). After addition of substrate from the kit, the membranes were read by the Typhoon imager, with ImageQuant software for resolution of images (Molecular Dynamics).3cells/well in microtiter plates in appropriate medium, as indicated. After 48 hr, 15 μl MTT  was added to each well for 4 hr, then the resulting formazan was dissolved in 0.1% SDS. Absorbance was determined at 570 nm on a Dynatech microplate reader. Statistical determinations of significance were performed by unpaired Student t-test for multiple independent assays, using GraphPad software.NRP-152, NRP-154, BPH-1, and transfected cells were seeded at 10The effect of dihydrotestosterone (DHT) as growth agonist, and the effect of flutamide (F) as growth antagonist, was assessed by use of the MTT assay described above. DHT and F were obtained from Boeringer-Mannheim, and cells were treated with 1 or both drugs at concentrations ranging from 1 to 100 nM for DHT, and 0.1 to 3 μM for F. These are within the published ranges of efficacy for these drugs ,54. Vehi32P-dCTP (New England Nuclear), then allowed to hybridize to the blot overnight in hybridization buffer. After washing, hybridization was detected by use of a PhosphoImager (Molecular Dynamics).Northern blot hybridizations to detect the retinoid receptors RARα, RARβ, and RARγ were performed as previously published . In brie6 cells; Caltag) for 15 min at room temperature. Apoptotic cells (cells staining with FITC-annexin V) were quantified by measuring green fluorescence in FL1 on the flow cytometer. In some experiments, cells were also stained with propidium iodide (PI), which is detected by the FL3 detector. CellQuest software was used to acquire and analyze the data on a Becton-Dickinson FACScan flow cytometer. For studies using the tyrphostin JAK2 inhibitor AG490 , the dissolved compound was added to subconfluent cells, as described [Forty-eight hr after transient transfection, cells were harvested using Enzyme-Free Cell Dissociation Buffer. After two washes with PBS, they were stained with FITC-annexin V (5 μl/10escribed . A vehic4 cells were trypsinized and washed in Ca2+/Mg2+-free PBS (Life Technologies) and plated in 1 ml of medium plus serum without supplements containing 0.3% (w/v) Noble Agar (Difco/Becton-Dickinson) over a 2 ml layer of the same medium with 0.6% agar in six-well plates. The number of colonies was counted using low magnification microscope (4×) after 10 days.Transfected cells were subjected for growth in soft agar to assess their change in phenotype with regards to colony formation. After selection and cloning, 106 S3c or vector-transfected (pIRES-EGFP) control cells. Each group consisted of 5 animals. In some experiments, the cells were mixed with Matrigel (Collaborative Research) prior to injection. Tumor growth was monitored weekly using engineer's caliper's to measure the 2 perpendicular diameters, over the course of 12 weeks.Our protocol was reviewed and approved by the Institutional Animal Care and Use Committee of UMDNJ. Severe-combined immunodeficient (SCID) mice (Charles River Laboratories) were obtained at 5 weeks of age, and acclimatized in the barrier vivarium for 1 week. At that time they were injected subcutaneously with 8 × 10STAT signal transducer and activator of transcriptioncSTAT3 constitutively-activated STAT3JAK Janus activated kinasePCA prostate cancerS3c constitutively-activated STAT3 gene having a Cys substitutionFLAG an immunogenic peptide fused to gene of protein to be expressed for identification and/or purification purposesSCID severe combined immunodeficientPBS phosphate-buffered salineDMEM Dulbecco's modifcation of Eagle's mediumIMDM Iscove's modification of Eagle's mediumFITC fluorescein isothiocyanatePE phycoerythrinPI propidium iodideFL1, 2, or 3 fluorescence detectors on a flow cytometer that collect fluorescence data within aset range of wavelengths, FL1 being the lowest and FL3 being the highestEGFP enhanced green fluorescence proteinRAR retinoic acid receptorDHT dihydrotestosteroneF flutamideHFH conceived of the retinoic acid receptor subunits experiments, and perofrmed the northern blot hybridizations. TFM performed the growth factor dependence and growth rate experments, performed some of the in vivo experiments, and prepared cells for flow cytometry. PS performed some of the transfections, participated in the in vivo experiments, the western blots, and prepared cells for flow cytometry. ABB made the pIRES-S3c plasmid from pBABE-S3c, and performed the soft-agar cloning experiments. BEB conceived of the project, performed most of the transfections, performed some of the in vivo experiments, and performed all flow cytometry acquisitions and analyses. All authors read and approved the manuscript.


Food-borne disease kills humans only rarely, although the ramifications and implications of these few deaths for science, regulators, and government are large When Food Kills: BSE, E. coli, and Disaster Science. The two food-borne diseases that occupy much of the book, Escherichia coli 0157 and bovine spongiform encephalopathy (BSE), kill humans very rarely, although the ramifications and implications of these few deaths for science, regulators, and government are large.For the estimated 800 million people, living largely in developing countries, without enough food to eat, the main food risk is starvation. But if you ask, ‘When does food actually kill?’ in a country such as the United Kingdom, ‘Not that often’ is the short reply you would give after reading Hugh Pennington's book E. coli 0157 kills fewer than a dozen people a year in the UK.As Pennington clearly explains, there is still much uncertainty in the science of BSE, and the eventual UK death toll from the human form may be as low as a few hundred, with even the most pessimistic expert assessments putting the upper bound as fewer than 5,000. Food-borne E. coli as food risks. For instance, a recent study by the King's Fund  reports that the rate of news coverage in the UK of a death from variant Creutzfeldt-Jakob disease, the human form of BSE, is nearly 23,000 times that for a death from obesity.Whilst each death is a terrible tragedy and an indescribably harrowing experience for those close to the victim, these figures are small when compared with other ways in which food kills. Epidemiologists estimate that the dietary contributions to cardiovascular disease and cancer between them kill more than 100,000 people a year in Britain. Yet we hear much more about BSE and In his characteristically diverting and obscurely erudite way, Pennington describes this discrepancy between public perception and magnitude of risk by referring to an article on railway accidents published in 1859 by one Dionysius Lardner. The systematic and much more revealing analyses of risk perception by psychologists such as Paul Slovic over the past 25 years do not get a mention.In fact, one of the hallmarks of Pennington's style is his enthusiasm for taking his reader down little-known historical byways. Whether it be the drowning (possibly suicide) of King Ludwig II of Bavaria in the Starnberger See or the treatment of James Norris in Bethlehem Lunatic Asylum in 1814, Pennington has an almost endless supply of anecdotes to provide peripheral colour to his main narrative. Indeed, on some occasions his delight in the detail makes it hard to see where the main narrative is leading, although his aim is to show that similar conclusions can be drawn about risk management in food, transport, oil rigs, and other fields.E. coli outbreak of 1996: ‘It was a stool. The word carries the impression of firmness, even of deliberate effort in its production. Hers was not’. His laconic sense of humour is also reflected in many of the wittily irrelevant or tangential photographs. My personal favourites are ‘Her Majesty in Gloves’ on page 44 and ‘Turds on Campsite Track’ on page 101.Anyone who has heard Hugh Pennington speak will know that he has a remarkably direct and engaging style, which he translates into the written word with verve. Already on page 2, he gets us into the mood by referring to a sample from a five-year-old girl sent for analysis at the start of the Lanarkshire E. coli 0157 outbreak, which in late 1996 affected 202 people and killed eight, was very much Pennington's show. He chaired the public enquiry that led eventually to a change in the law, requiring all butchers in the UK handling cooked and raw meat to be licensed. The license itself is less important than the training in food safety management principles that precedes it. The butcher John Barr (and his staff), whose shop was the primary source of the outbreak, apparently did not know that you have to keep raw meat and ready-to-eat products separate to avoid cross-contamination with dangerous pathogens, such as E. coli 0157, that can occur in raw meat. Pennington's authoritative and blow-by-blow account shows failings not only in the butcher , but also in the inspectors who had visited his shop eight times in the previous two years. They had not, apparently, picked up that Barr and his staff employed the same knives for cutting up raw and cooked meat, nor that they used a ‘biodegradable’ cleaning fluid, not realising that this is not the same as ‘biocidal’.The Lanarkshire The second theme, BSE, is given somewhat shorter treatment. Nevertheless, Pennington goes into some detail in assessing the prion theory of transmissible spongiform encephalopathies . He also reviews the sequence of events that led the UK government in the early 1990s to conclude that there was not likely to be a risk to human health and to be slow to change its view. This and the concluding part of the book (see below) draw heavily on the Phillips Enquiry into BSE. Although this enquiry focussed on the response of the UK government, its lessons are relevant to other countries where BSE has emerged in recent years, including many European countries, Japan, Canada, and the United States.Mountains of the Mind, Robert Macfarlane writes: ‘[F]or the hunter risk wasn't optional—it came with the job. I sought risk out, however. I courted, in fact paid for it. This is the great shift which has taken place in the history of risk…. [I]t became a commodity’. Pennington reflects a similar shift in attitude to food risk over the past half century or so. Back in 1938, although it was known that over 2,500 people a year in Britain died from drinking raw milk, the risk was not seen as large enough to warrant legislation to make pasteurisation compulsory. We are now used to much higher standards of food safety, and we can, as a society, enjoy the luxury of fear of relatively minor risks.In his book Nevertheless, there are important lessons from past failures for all involved in food safety (and in other areas of risk management), and Pennington discusses some of these in his concluding chapters. He emphasises the need to continually review the evidence underpinning risk assessments, to communicate effectively with the media, to ensure that actions to manage risks are effectively implemented and audited. Notably, he refers to the importance of inclusiveness and openness about risk and uncertainty in decision-making: ‘[I]f [this] becomes the norm, it will be possible to say that good has come out of tragedy’.


Isoflavones from soybeans may provide some beneficial impacts on postmenopausal health. The purpose of this study was to compare the pharmacokinetics and bioavailability of plasma isoflavones (daidzein and genistein) after a single dose of orally administered soy beverage and soy extract capsules in postmenopausal Thai women.max), time to maximal plasma concentration (Tmax), area under the plasma concentration-time curve (AUC) and half-life (t1/2), were estimated using the TopFit version 2.0 software with noncompartmental model analysis.We conducted a randomized two-phase crossover pharmacokinetic study in 12 postmenopausal Thai women. In the first phase, each subject randomly received either 2 soy extract capsules (containing daidzin : genistin = 7.79 : 22.57 mg), or soy beverage prepared from 15 g of soy flour (containing daidzin : genistin = 9.27 : 10.51 mg). In the second phase, the subjects received an alternative preparation in the same manner after a washout period of at least 1 week. Blood samples were collected immediately before and at 0.5, 1, 2, 4, 6, 8, 10, 12, 24 and 32 h after administration of the soy preparation in each phase. Plasma daidzein and genistein concentrations were determined by using high performance liquid chromatography (HPLC). The pharmacokinetic parameters of daidzein and genistein, i.e. maximal plasma concentration  higher than those of soy beverage.There were no significant differences in the mean values of Cmax adjusted for the dose, Tmax and t1/2, were not different between both soy preparations.The bioavailability of daidzein, which was adjusted for the administered dose (AUC/dose), following a single oral administration of soy beverage was slightly (but significantly) less than that of soy extract capsules, whereas, the bioavailability adjusted for administered dose of genistein from both soy preparations were comparable. The other pharmacokinetic parameters of daidzein and genistein, including C Menopause is associated with estrogen deficiency and its accompanying symptoms such as accelerated bone loss and atherosclerosis. Hormone replacement therapy (HRT) has traditionally been used for treatment of menopausal disorders. Estrogen helps to maintain bone density, relieve menopausal symptoms, as well as influence emotional state . EstrogeThe most common forms of phytoestrogens are the isoflavones. Among the food consumed by humans, soybeans contain the highest concentration of isoflavones. Isoflavones have a chemical structure resembling that of estrodiol-17β, the most potent mammalian estrogen. The major isoflavones, namely, genistein and daidzein, have several features in common with estradiol-17β, including an aromatic A ring with hydroxyl group in the same plane at a distance similar to that in estradiol , cardiovascular diseases and osteoporosis -11. ThusAlthough many commercial soy capsules containing isoflavone extract are now available in many Western countries, soybeans and soy food (such as tofu, soy flour, soy milk, etc.), which provide the main sources of isoflavones, are consumed in significant amounts in Asian countries because they are inexpensive and high in quality protein. The purpose of this trial was to compare the pharmacokinetics of plasma isoflavones, daidzein and genistein, in postmenopausal Thai women after a single dose of orally administered commercial soy extract capsules and soy beverage.This study was a single dose, randomized two-phase crossover study with a washout period of at least one week. It was approved by the Medical Ethics Committee of the Faculty of Medicine, Chiang Mai University and was in compliance with the Helsinki Declaration.2 (22.06 ± 1.83 kg/m2). Their serum follicle-stimulating hormone concentrations were more than 20 IU/l and the average level was 69.60 ± 31.21 IU/l. All had to be in good health on the basis of medical history and physical examination. Routine blood tests including complete blood count (CBC) with differential, blood urea nitrogen (BUN), creatinine (Cr) and liver function test (LFT) had to be within the normal limit. Subjects had to give both verbal and written information regarding the study. Signed informed consent was obtained prior to entry. Exclusion criteria included subjects with known premenopausal status  as well as those with a known history of chronic renal, liver, pulmonary or cardiovascular diseases, recent cigarette smoking, substance abuse or addiction, use of antibiotics within the previous 6 weeks, consumption of more than 2 alcoholic drinks/day, regular use (more than 1 dose/week) of over-the-counter or prescribed medications, and malignancy.A total of 12 postmenopausal Thai women, who ranged in age from 46–61 years (average 52.83 ± 3.88 years), participated in this study. Their mean weight and height was 52.23 ± 6.38 kg and 1.54 ± 0.06 cm, respectively. The body mass index (BMI) of each subject was within 18–24 kg/m®, Chiang Mai, Thailand, lot no. 19GODE) with 300 ml of hot water. The concentration of isoflavones in each preparation was measured as aglycones (daidzein and genistein) and β-glycoside conjugates of isoflavones (daidzin and genistin) that were analyzed by high performance liquid chromatography (HPLC) as described below.The isoflavone preparations used in this study were commercial soy extract capsules and soy beverage. Soy extract capsules were purchased from Canada, whereas, the soy beverage was prepared from mixing 15 g of commercial soy flour . After the blood sample collection at 12 h postdose, the subjects were discharged from the Clinical Pharmacology Unit and asked to come back again on the next day to give blood samples at 24 and 32 h postdose. While waiting for blood sample collections, the subjects were allowed to perform all of their daily activities, except moderate to high degrees of exercises. After a washout period of at least 1 week, the subjects received the alternative preparation and the blood samples were collected in the same manner. Identical food and fluid were served during the 2 study periods. The subjects were required to refrain from drinking caffeine containing beverages and alcohol, and instructed to consume no soy products (except those given in this study) from the time of screening until the end of the research.®) and collected in a heparinized vacutainer (BD Insyte®). The blood collecting tubes were centrifuged at 2,500 rpm for 20 min and the plasma samples were separated and frozen at -20°C until analyzed.Venous blood samples (7 ml/each) for determination of soy isoflavones were collected predose and then exactly 0.5, 1, 2, 4, 6, 8, 10, 12, 24 and 32 h after administration. Samples were obtained from the forearm by venipuncture through an indwelling intravenous catheter (BD InsyteHelix pomatia (Sigma G-0876) to hydrolyze glucuronide and sulfate conjugates of genistein and daidzein. The enzyme mixture was made up freshly and contained 0.15 g of ascorbic acid in 10 ml of 0.2 M acetate buffer, pH 4.0, and 500 μl of β-glucuronidase/sulfatase from Helix pomatia. To allow for complete hydrolysis in the plasma samples, 0.75 ml of 0.2 M ammonium acetate buffer was added, and the tubes were capped and heated overnight in a water bath (15–18 h, 37°C). The tubes were removed from the water bath and allowed to cool to room temperature. After enzymatic hydrolysis, plasma samples were spiked with 5 μl of internal standard (IS, 100,000 ng/ml fluorescein in 80% methanol). After vortex mixing for 30 sec and centrifugation at 13,000 rpm for 5 min, sample purification was performed by using a solid phase extraction cartridge placed in the vacuum box. The cartridges were preconditioned with 2.50 ml each of methanol, water and 175 mM phosphate buffer, respectively. The samples were loaded into the cartridges and allowed to flow freely, then the cartridges were washed with 0.1 ml of 20% methanol in water, 0.1 ml of 175 mM phosphate buffer and 0.1 ml of 20% methanol in water, consecutively. After drying the cartridge by slow suction, isoflavones and IS were eluted with 2.0 ml of 20% methanol in ethyl acetate. The eluents were dried by a SpeedVac concentrator. The residues were dissolved in 50 μl of the mobile phase, and 10 μl of samples were injected into a HPLC system. The mobile phase consisted of 4 mM perchloric acid in methanol/acetonitrile (115:85, v/v). The flow rate was maintained at 1 ml/min and the analytes were detected by UV absorption at 259 nm, and the column was maintained at 40°C. The retention time for daidzein and genistein was 7.699 and 13.031 minutes, respectively. The lower limit of quantitation was 5 ng/ml. Plasma concentrations of daidzein and genistein were determined using a calibration curve and linear regression of the seven known isoflavone concentrations versus the peak height ratios of isoflavones and IS.The assay was modified from the solid phase extraction procedure and HPLC technique as previously described by Thomas et al . BrieflyThe %CV of intraday precision for plasma daidzein concentrations ranged from 0.85–9.45%, whereas, the %CV of interday ranged from 3.90–9.96%. The %CV of intraday and interday precision for plasma genistein concentrations ranged from 1.29–3.65%, and 4.37–6.84%, respectively. The %deviation in intraday and interday assay for plasma daidzein concentrations ranged from 4.35–5.84% and -6.11–4.22%, respectively. On the other hand, the %deviation in intraday and interday assay for plasma genistein concentrations ranged from -5.00–1.98% and -7.79%–1.52%, respectively.max, ng/ml) and time to maximal plasma concentration (Tmax, h) were obtained directly by the visual inspection of each subject's plasma concentration-time profile. The areas under the plasma concentration-time curve from time 0–32 (AUC0–32, ng.h/ml) and 0-∝ (AUC0-∞, ng.h/ml) as well as half-life (t1/2, h) were determined by non-compartmental analysis. The slope of the terminal log-linear portion of the concentration-time profile was determined by least-squares regression analysis and used as the elimination rate constant (Ke). The elimination t1/2 was calculated as 0.693/Ke. The AUC from time zero to the last quantifiable point (AUC0–32) was calculated using the trapezoidal rule. Extrapolated AUC from time t to infinity (AUCt-∞) was determined as Ct/Ke. Total AUC was the sum of AUC0–32+ AUC32-∞. In this study, the sampling time was continued for more than 3 half-lives, therefore, the AUC0-32 was sufficient to cover at least 80% of the total AUC. The calculation was performed by using the TopFit software version 2.0 for personal computer.The maximal plasma concentration  or Wilcoxon's sign rank test (non-parametric method).The pharmacokinetic parameters were presented as mean ± SD. The differences between the mean values of CThe quantification of isoflavone contents demonstrated that both soy flour and soy extract capsule preparations contained predominantly daidzin and genistin (the form of β-glycoside conjugates), whereas, aglycones were rarely found and isoflavones in other forms were not measured. Isoflavone contents in both soy preparations are shown in Table The Mean plasma daidzein and genistein concentration-time curves after a single dose of both orally administered soy preparations are shown in Figure max) were 96.31 ± 36.18 ng/ml and 96.02 ± 27.71 ng/ml for soy beverage and soy extract capsules, respectively. Pharmacokinetic analysis of the plasma concentration-time curves showed that the elimination t1/2 was 7.68 ± 4.14 h for soy beverage and 6.67 ± 1.65 h for soy extract capsules. The AUC0–32 was 996.86 ± 455.69 and 1110.94 ± 342.26 ng.h/ml for soy beverage and soy extract capsules, respectively, whereas, the AUC0-∝ was 1,150.73 ± 504.94 ng.h/ml for soy beverage and 1,211 ± 354.46 ng.h/ml for soy extract capsules.The mean plasma daidzein concentration-time curve after a single dose of both orally administered soy preparations revealed a biphasic pattern. The first peak of plasma daidzein concentration was reached approximately 1 h after ingestion of both preparations, whereas, the second peak attained higher plasma concentrations at 5.92 ± 2.43 h for soy beverage, and 6.25 ± 2.26 h for soy extract capsules. The mean maximal plasma daidzein concentrations  higher than those after soy beverage intake  as well as daidzin and genistin content (9.1 mg/capsule, each) were declared. Nonetheless, our quantification of isoflavones in this preparation revealed that the amount of daidzin and genistin in each capsule was 3.90 ± 0.04 and 11.29 ± 0.17, respectively. Therefore, the orally administered dose in this study was calculated according to our quantification, but not by the amount declared.Initially, we tried to measure the total isoflavone contents in each preparation by using the acid hydrolysis method ,15. BrieSince both soy preparations consist of different proportions of daidzin : genistin (approximately 1:1 for soy flour, 1:3 for soy extract capsules), the appropriate amount of daidzin content in each preparation was therefore considered first for pharmacokinetic comparison. In this study, each volunteer was assigned to receive 2 capsules of soy extract (daidzin : genistin = 7.79 : 22.57 mg) to compare with soy beverage prepared from 15 g of soy flour (9.27 : 10.51 mg). These dosages caused an approximately equal amount of daidzin between the two preparations, whereas, the genistin content in soy extract capsules was approximately two-fold higher than that of soy beverage. The oral administration of these dosages resulted in the plasma concentrations of daidzein and genistein being high enough and convenient for measurement by the HPLC method. In addition, preparing soy beverage from 15 g of soy flour in 300 ml of water was practical and created an acceptable concentration for consumption.0–32/dose and AUC0-∝/dose) of genistein after ingestion of both soy preparations, was not significantly different. In contrast, the bioavailability of daidzein following ingestion of soy extract capsules was significantly greater than that following ingestion of soy beverage. This might be the result of at least 2 possibilities. Firstly, the food matrix of soy flour may alter the bioavailability of daidzein. Dietary factors such as fiber and carbohydrate have been associated with differences in the metabolism of daidzein to equol [From previous studies, the bioavailability of isoflavones was investigated and compared among various soy food and beverages. So far, there has been no study that compares bioavailability of isoflavones from soy extract versus natural soy food or beverages. Our purpose was to investigate pharmacokinetics of daidzein and genistein after ingestion of soy beverage compared to soy extract capsules in postmenopausal Thai women. Daidzein and genistein contents in soy food can vary and depend on the raw material and processing conditions used to produce a particular food product. Furthermore, in each type of soy food, there are different forms of isoflavones in differing amounts. However, based on the equivalent dose of isoflavones, the administration of different soy food has shown no difference in isoflavone bioavailability . In our to equol -20. Urinmax of daidzein and genistein from both soy preparations in this study was shorter than the values of 8–11 h (after ingestion of β-glycoside conjugates) as reported in previous studies [1/2 of daidzein and genistein in this study was comparable to the values of 6–8 h from other studies [The mean T studies ,22. This studies -22.It has been suggested that a daily intake of soy isoflavone extract containing 50/50 mg of genistin and daidzin  or 76 mgmax adjusted for dose, Tmax and t1/2 between both soy preparations.The bioavailability of daidzein, which was adjusted for the administered dose (AUC/dose) following a single oral administration of soy beverage, was slightly (but significantly) less than that of soy extract capsules, whereas, that of genistein from both soy preparations was comparable. There was also no difference in other pharmacokinetic parameters of daidzein and genistein, including CThe author(s) declare that they have no competing interests.EA performed the quantification of isoflavones and statistical analysis. ST supervised data collection and analysis, and drafted the manuscript. NR supervised the quantification of isoflavones. SP initiated the research question and participated in the selection of patients eligible for the study. CS participated in the design of the study and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


To compare patterns of use, beliefs, and attitudes related to waterpipe smoking between university students (beginning smokers) and café customers (established smokers) in Aleppo Syria, in order to explore the evolution of this smoking method.Two cross-sectional surveys were conducted among representative samples of university students , and waterpipe users among cafe' customers  in Aleppo, Syria. We used interviewer-administered questionnaire inquiring about pattern of waterpipe smoking (initiation, frequency), situational characteristics of use (partner, place, sharing), beliefs related to waterpipe smoking (harmful/addictive properties of waterpipe), attitudes related to waterpipe smoking (confidence in quitting, will to quit, motivation for quitting, past year quit attempt), and cigarette smoking.Daily and regular patterns of smoking become more prevalent with increased duration of smoking, but intermittent smoking remains the predominant pattern of waterpipe use. Women seem to be drawn later to the habit, which seem to escape the usual taboo against women's cigarette smoking. Patterns and context of waterpipe use tend to change with progress of the practice affecting frequency, setting, and sharing of waterpipe. Unlike beginners, established waterpipe smokers seem more smoking-method oriented, more hooked on the habit, less willing to quit, and less likely to foresee challenges to quitting.Use patterns and attitudes related to waterpipe smoking evolve to accommodate the change in dependence and life circumstances of the smoker. Most of use features, beliefs, attitudes, as well as time-course seem unique to this smoking method requiring novel approach to intervention. Waterpipe, is a generic name for tobacco use methods that share a common feature; passage of smoke through the water before inhalation. It is known under different names and shapes in different cultures and countries (e.g. shisha, narghile, hookah, hubble bubble) . AlthougStudies on the health effects of this smoking method are scanty, and often suffer from poor control of other confounding factors (e.g. cigarette smoking). We are just beginning to learn in a more methodological way the full spectrum of harmful effects of this smoking method and about shared and distinguishing features from cigarette smoking. For example, in regards to CO, one of the main cardiovascular risks of cigarette smoking, waterpipes are at least as efficient as cigarettes in delivering this toxic gas to smokers' lung . On the Because of the increasing trend of this smoking method, together with its health damaging and addictive potentials the need to develop ways to intervene with smokers and prevent initiation seems timely. Intervention efforts to deal with this emerging public health problem however, are hindered by the inadequacy of data characterizing various aspects related to patterns of waterpipe use and dependence .We recently conducted a survey looking at prevalence, beliefs, and attitudes related to this smoking method among university students in Aleppo, Syria. Waterpipe was practiced by a quarter of male and 5% of female students and was characterized by intermittent and social use . Also, sThe methods of these surveys conducted in 2003 are described in details elsewhere . BrieflyThe questionnaires used in these surveys were developed from standard instruments modified to suit waterpipe smoking -13. The Generally, the two groups were compared on the following dimensions; 1- socio-demographics including age, gender, number of years of formal education, economic status assessed by the density index (DI = number of household members/number of rooms in the house), 2- smoking characteristics including frequency of use , age of initiation of use, age of initiation of daily use, cigarette smoking status (past month cigarette smoking, ex-daily smoking for current non-smokers), 3- situational characteristics of waterpipe use including waterpipe smoking initiation , current waterpipe smoking , usual place of waterpipe smoking (open end question categorized later into home/dorms, cafes, other or no particular place), waterpipe sharing with others, usual sharing companion (as an open end question categorized later into friend, family), whether use is periodic/seasonal, the season of increased use (as an open end question categorized later into holidays, summer/spring, stress/exams, and other), 4- quit attitude (waterpipe, cigarette) including belief of own ability to quit, will to quit, past year quit attempt, quit motivation , perceived major challenge for quitting , 5- perceived main health hazard of waterpipe smoking (open end question categorized into cardiovascular effects, respiratory effects, cancer, other bodily effects, none or don't know), harmful and addictive effects of cigarettes compared to waterpipe , and finally 6- family's attitude towards waterpipe and/or cigarette smoking categorized into , and smokers' perception of the extent they are hooked on waterpipe  , and were compared between the two groups using Student's t test or the Mann-Whitney test as appropriate. Spearman correlation coefficient was calculated for the relation between duration of waterpipe smoking and frequency of use (categorized as daily, weekly, monthly) in the café study. SPSS statistical software (release 11) was used for the analysis with p value <0.05 considered significant.Frequency analyses of main study indices were tabulated and differences between the two groups were assessed using the Chip = 0.02). Mean duration of waterpipe smoking session among café customers was 71.1 ± 35.8 minutes.Overall, the two samples combined show that daily waterpipe smoking was seen among 19.5% of participants (23.3% of smoking men and 12.3% of smoking women), while occasional smoking was seen among 80.2% of participants (76.3% of smoking men and 87.7% of smoking women). More than half of waterpipe users initiated use and currently use waterpipe with a friend (Table p < 0.01), still ex-cigarette smoking did not differ between the two groups. Students as well were more likely to have a seasonal pattern of waterpipe use, which was more associated with exams and stress compared to café customers (Table Comparison of patterns of initiation and current use of waterpipe between café customers and university students is detailed in Table rs Table .Quit attitude of café customers shows that the majority (86.5%) believe that they can quit waterpipe any time, but only a minority (28.4%) are interested in quitting, mainly out of health motivation Table . In compp < 0.001, p = 0.1, respectively).Attitude of families towards participants' waterpipe smoking for both café customers and students are depicted in figure p < 0.01 for both).Finally, both mean education years and DI differed significantly between café customers  and students (mean education years 14.7 ± 1.4, mean DI 2.0 ± 0.7)  among café customers. Still, unlike cigarette smoking, which is practiced mainly on a daily basis in the two studied populations and in the Syrian society in general , intermiAge of initiation of waterpipe differed significantly between men and women for both studied groups . This may explain why the proportion of women among café customers is double that of students, although one should bear in mind the limitation of the samples for such a comparison. We have to be mindful as well that unlike cigarette smoking we are still at the expansion stage of the waterpipe epidemic, where people of different age groups are joining the new hype at relatively the same time period. An indication of this fact is that while age of initiation of waterpipe differed between the two studied groups, age of initiation of daily cigarette smoking was somehow identical. The tolerant attitude towards waterpipe smoking by women can aid its spread among them Figure  3,15]. ,15. 3,15The social dimension, which is a salient feature of waterpipe smoking, seems also to differ between the two studied groups. So while students mainly initiate and currently practice the habit with their friends, more smokers among café customers are initiating or currently practicing the habit with family members or alone, despite being sampled in a social setting. It appears that the social context of waterpipe smoking changes with the change of both personal and smoking related characteristics (marriage, increased dependence). Our previous analysis of factors related to frequency of waterpipe use, as a marker of dependence, has lead us to suggest that as waterpipe smokers become more dependant the social pattern of smoking is gradually replaced with more individual one . SharingWaterpipe quit attitude and perceived challenges to quitting differed between café customers and university students, with café customers demonstrating lower interest in quitting and lower perception of challenges to quitting Table . This caAs mentioned above a limitation of this study is that the café sample cannot be considered representative of adult smokers in the community. Also, the study consists of two cross sectional surveys in two populations presumably at two different stages of their waterpipe practice. Still, the two groups are not totally distinct from each other as they overlap in terms of age and smoking characteristics. However, as noted from the discussion we are mindful of these limitations throughout analyses and interpretation of the study's results.This study shows the predominance of intermittent use of this smoking method as well as its increasing popularity among women who are drawn to the habit relatively later than men. The social context of waterpipe smoking, which is a defining feature of this smoking method, tends to change to accommodate smoker's life and dependence progress. Our data suggest that at early stages of waterpipe use, users tend to come from cigarette smokers or people with liberal attitude towards smoking in general, but at more advanced stages of use smokers tend to be more smoking method-oriented and less keen on quitting. There is a general belief of easiness to quit waterpipe compared to cigarette, but this is likely to be an under-estimation as it is not translated into high success rate of quit attempts. Intervention or prevention strategies to curb this emerging epidemic should take into consideration the unique use features of this smoking method as well as smokers' perceptions and attitudes towards it.The author(s) declare that they have no competing interests.TA designed the study and wrote the first draft. KDW and TE participated in the study design and co-authored the manuscript. WM participated in the study design and wrote the final draft.The pre-publication history for this paper can be accessed here:


Many recent studies describe learning-related changes in sensory and motor areas, but few have directly probed for improvement in neuronal coding after learning. We used information theory to analyze single-cell activity from the primary motor cortex of monkeys, before and after learning a local rotational visuomotor task. We show that after learning, neurons in the primary motor cortex conveyed more information about the direction of movement and did so with relation to their directional sensitivity. Similar to recent findings in sensory systems, this specific improvement in encoding is correlated with an increase in the slope of the neurons' tuning curve. We further demonstrate that the improved information after learning enables a more accurate reconstruction of movement direction from neuronal populations. Our results suggest that similar mechanisms govern learning in sensory and motor areas and provide further evidence for a tight relationship between the locality of learning and the properties of neurons; namely, cells only show plasticity if their preferred direction is near the training one. The results also suggest that simple learning tasks can enhance the performance of brain–machine interfaces. Information theory is used to analyze neuronal responses in monkeys trained to direct movements to a visual target Practice can induce behavioral improvement that is often specific to the situation experienced during the practice sessions (or “training”). Such findings suggest that changes occur in neurons with fine selectivity (or “tuning”) for the stimuli experienced or the movements made during training. In the visual system, for example, behavioral improvement is specific to the trained stimulus, such as the orientation of a light bar , and is Whereas many studies indicate that learning can induce specific changes in brain activity, this finding does not necessarily imply that newly learned skills are “better” represented in the brain. The crucial question is this: Do neurons encode task parameters, such as movement direction, any better after learning? In the motor system, such improved encoding  can be uTo address the first question, we employed an information-theory analysis  to calcuMonkeys adapted to visuomotor rotations on a daily basis by altering the relationship between the visual feedback (cursor) and the hand movement . LearninActivity was measured from the hold period that immediately follows the target appearance, but before the go-signal, and was therefore termed preparatory activity (PA). There were three reasons for this choice. First, such PA has been reported in many motor cortices and is thought to participate in movement planning and in computing visuomotor transformations . Second,p = 0.3) or by comparing their means (paired t-test, p = 0.53). We further tested the average information about direction conveyed by each spike by normalizing each cell's information by its firing rate and again found no significant difference (inset in p = 0.25, paired t-test, p = 0.7).The MuI between one-cell activity and direction of movement is exemplified in p-value. A high p-value means that the MuI in the post-learning epoch was significantly higher than the MuI in the pre-learning epoch. p-values for all cells. A significant number of cells showed an increase in MuI with a p-value larger than 0.95 (black in n = 37 out of 177, p < 0.01, χ2), a nearly significant number of cells showed a decrease in MuI with a p-value lower than 0.05 (white/transparent in n = 18, p = 0.06), while all the rest did not  was compared to this distribution to obtain a  gray in B. We als gray in C, upper  gray in C, lower.p < 0.05) with its center on the learned direction. In contrast, the PD distributions of the whole population  in local rotational transformations and the sensitivity of many cells to direction, we probed for a relation between cells' PD and the learned direction. p > 0.1, χ2) number of cells (eight out of 126) had a p-value greater than 0.95 (p > 0.1).To test that this change in information is indeed owing to the learning of visuomotor transformations and not owing to the mere repetition of a single movement during the learning epoch, we conducted the same analysis for control, repetition sessions. Only a nonsignificant   and is mp = 0.01) was found with its peak on the learned direction  stimulus–response distribution and thus does not tell us what feature in cell activity primarily contributed to the increase in information content. However, we found that the increase in information is specific to a single-learned direction and that cells with a PD close to the learned direction mainly contributed to this increase. We have previously reported that cells with PD close to the learned direction increased their firing rate after learning when movement was to the learned direction . We therTwo natural features of a cell's tuning curve can contribute to the improvement in information content. First, a cell can increase the slope of the tuning curve just near the learned direction, and thus small changes in direction can lead to a larger difference in the cell's response, providing a better differentiation of direction based on the neuronal activity  approach, a widely used decoding scheme for M1 activity , and a mp < 0.05 for all four learned directions, bootstrap and t-test). We verified that this improvement was due to learning in two ways: first, by shuffling trials from the pre-learning and the post-learning epochs, and second, by shuffling cells from days with different transformations. In both cases, the mean of the distribution of improvements was not significantly different from zero. Furthermore, the improvement in the PV prediction was specific to the learned-movement direction. 2, p < 0.01). This improvement in the PV prediction can be accounted for by the enhanced firing of cells with a PD near the learned-movement direction, as shown above  was similar for our monkeys  and in hAn intriguing result in this study is that learning-related changes were observed and persistent in the post-learning epoch, when performing a standard task that required no transformation. Further, measured kinematics was the same as in the pre-learning epoch. If the improved information can be used, why isn't it? First, our monkeys were trained on a task that did not require improved performance in the standard task after learning, but did encourage them to reserve learning for future use of the same visuomotor task. This is in agreement with our previous report, showing that the monkeys retained the task until the performance of the relearning epoch , and suggests that the neuronal change should persist but somehow gated. Indeed, everyday behavior shows that we can learn new tasks without interfering with the performance of existing ones. An alternative possibility is that we did not measure the appropriate kinematic variable that was altered and improved due to the neuronal changes. For example, a task that would demand finer directional sensitivity (i.e., angular distance of less than 45°) might show a change in performance after learning.It is also worth noting that our experiment was performed in a local region in space and was not constrained to a specific posture  or jointOne important question is what kind of learning can induce such an increase in information content. Although psychophysics studies have shown that adapting to new kinematics and/or dynamics environments results in the formation of internal representations in the brain  monkeys (approximately 4.5 kg) were implanted with recording chambers (27 × 27 mm) above both the right and left hemispheres. Animal care and surgical procedures complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (rev. 1996) and with the Hebrew University guidelines supervised by the Institutional Committee for Animal Care and Use. The monkeys were seated in a dark chamber, and eight microelectrodes were introduced into each hemisphere. The electrode signals were amplified, filtered, and sorted (MCP-PLUS, Alpha-Omega, Nazareth, Israel), and all spike shapes were sampled at 24 KHz. We used a template-based method for real-time isolation of spike shapes (MSD, Alpha-Omega).Two female rhesus (Penetration locations were verified by MRI (Biospec Bruker 4.7 Tesla, Bruker BioScences, Billerica, Massachusetts, United States) before recordings. At the end of each session, we examined the activity of neurons evoked by passive manipulation of the limbs and applied intracortical microstimulation  to evoke movements. Only penetration sites that evoked single-joint shoulder or elbow movement at thresholds of lesser than or equal to 40 μA were used in this study. In one monkey, we also made anatomical observations, to verify the accurate penetration sites relative to the central sulcus.learned-movement direction refers to the direction of hand movement needed to bring the cursor to the target for these rotations (thus, there were four possible learned-movement directions in this study: 0°, 45°, 135°, and 180°, associated with the –90°, –45°, +45°, and +90° transforms, respectively). Monkeys were trained for several months with the standard eight-target, task but did not see the transformations before the recordings. To achieve learning on a daily basis during the whole recording period (rather than switching between pre-learned behaviors), a different rotational transformation was randomly chosen for each day from the set of four possible transformations. To observe systematic change in the activity of neurons, the same transformation was repeated .Monkeys moved a manipulandum to control the movement of a cursor on a video screen located 50 cm from their torso and eyes with the goal of moving the cursor from a starting point at the center of the screen (origin) to a visual target in a delayed go-signal paradigm; this required the monkey to hold (as verified by hand velocity and EMG) the cursor in the origin circle for a random 750–1,500 ms after the target onset. The disappearance of the origin indicated the go-signal. In each session (day), four consecutive epochs were introduced: (1) pre-learning epoch , a standard, eight-target task in which the target direction was randomly chosen from eight possible directions uniformly distributed over the circle; (2) learning epoch , a transformed, one-target task in which only one target (upwards, 90°) was presented and a rotational transformation was introduced between the cursor on the screen and the manipulandum; (3) post-learning epoch , where the default eight-target task was presented again; and (4) relearning epoch, same as the learning epoch. Rotations were 90°, 45°, –45°, or –90° and were chosen randomly for each session, but fixed for the duration of the learning epoch in a session. Note that learning here is local in direction since only one target direction was introduced during the learning epoch. The term Note that in this paradigm, the monkeys learn the visuomotor rotation by repeated performance of a single movement (to the learned direction). To test whether the repetition could be responsible for the neuronal changes observed, we conducted control sessions. These sessions (termed “‘repetition”' sessions) consisted of a one-target task without any visuomotor transformation (namely, a standard task to one direction only). We performed 16 such sessions (ten with repeated movements to 90° and six with movements to 180°).t-test, p > 0.1).Psychophysics studies have shown that immediately after learning, humans exhibit aftereffects, which is evidence for the formation of an internal representation of the newly acquired skill . This haWe also verified that learning was the same during the whole recording period. We divided the recording period into two to three consecutive segments and compared (1) learning rates in the learning epochs and (2) aftereffect magnitudes and washout rates in the post-learning epoch .U-test); (2) the results of a one-way ANOVA showing a significant effect for direction; (3) more than five trials per direction both pre- and post-learning. We calculated spike counts in the 600-ms range following the target onset, referred to as the PA. Examining the neurons for changes in PD did not reveal any systematic or significant changes (bootstrap test, three of 177 showed a significant change) and PDs were uniformly distributed (Rayleigh test).To further avoid changes in activity that result from any kinematic or dynamic differences, and since learning-related changes were only observed in activity taken from preparation for movement , here we only report neuronal activity from this period, i.e., activity during the 600 ms following the target appearance but before the go-signal. We isolated 177 cells (113 from monkey W and 64 from monkey X) based on (1) the lack of significant change in activity during the first-hold period  for the pre-learning epoch versus the post-learning epoch  or by assuming a Poisson distribution with the mean taken from all trials. We compensated for the limited number of trials (bias correction) by applying either analytical correction  MAP estimator. This was carried out by assuming Poisson distribution of rates and independency between neurons. We sampled (with repetition) 100 cells and then cross-validated by selecting randomly one trial from each cell and direction, calculating the cell's mean firing rate from the rest of the trials and used the following formula  to obtaii(d) denotes the mean firing rate of cell i in direction d and ir is the rate in the randomly drawn trial. For ease of computation, we took the log of the probability and did not calculate N, the normalization factor. The process was repeated 100 times and performed separately for the pre-learning and post-learning.where σ2r > 0.5). The cells' PDs were homogenously distributed both pre- and post-learning (Rao test, pre-learning, p = 0.4, post-learning, p = 0.5). We performed two bootstrap tests for significance. First, we shuffled trials from pre- and post-learning and calculated the difference between the deviations of the PV prediction pre-learning to that of the post-learning. The process was repeated 1,000 times to obtain confidence intervals. Second, we shuffled cells from days in which different transformations were learned and again obtained confidence limits. This process tests whether the improvement in prediction was indeed related to the specific direction learned in the session.(2) PV analysis . One hun