Phosphate overaccumulates in shoots in response to Zn deprivation. Results shown in this article suggest key roles of PHR1 and PHO1 and a counteractive function of PHO1;H3 in controlling root-to-shoot phosphate translocation in Arabidopsis. Arabidopsis thaliana and uncovering genes involved in the Zn–Pi synergy. Wild-type plants as well as mutants affected in Pi signalling and transport genes, namely the transcription factor PHR1, the E2-conjugase PHO2, and the Pi exporter PHO1, were examined. Zn deficiency caused an increase in shoot Pi content in the wild type as well as in the pho2 mutant, but not in the phr1 or pho1 mutants. This indicated that PHR1 and PHO1 participate in the coregulation of Zn and Pi homeostasis. Zn deprivation had a very limited effect on transcript levels of Pi-starvation-responsive genes such as AT4, IPS1, and microRNA399, or on of members of the high-affinity Pi transporter family PHT1. Interestingly, one of the PHO1 homologues, PHO1;H3, was upregulated in response to Zn deficiency. The expression pattern of PHO1 and PHO1;H3 were similar, both being expressed in cells of the root vascular cylinder and both localized to the Golgi when expressed transiently in tobacco cells. When grown in Zn-free medium, pho1;h3 mutant plants displayed higher Pi contents in the shoots than wild-type plants. This was, however, not observed in a pho1 pho1;h3 double mutant, suggesting that PHO1;H3 restricts root-to-shoot Pi transfer requiring PHO1 function for Pi homeostasis in response to Zn deficiency.Interactions between zinc (Zn) and phosphate (Pi) nutrition in plants have long been recognized, but little information is available on their molecular bases and biological significance. This work aimed at examining the effects of Zn deficiency on Pi accumulation in  Plants require phosphorus (P) and zinc (Zn) to ensure various basic biological functions and complete their life cycle . Plants Arabidopsis, a recent report by PHT1;1 in roots and shoots, respectively.Zn concentration in wheat and maize plants has been reported to decrease with the application of P, while Pi concentration decreases with the application of Zn . In barl+-Pi cotransporters (PHT)  is characterized as a component of the shoot-to-root Pi-starvation signalling cascade . Numerous observations reported the involvement of the low-molecular-weight chelator nicotianamine in the regulation of Zn homeostasis in plants  reporter gene  and introduced into Arabidopsis ecotype Col-0 as previously described  were obtained from the European Arabidopsis Stock Centre (www.arabidopsis.info) . The complete nutrient medium contained 0.5mM KNO3, 1mM MgSO4, 1mM KH2PO4, 0.25mM Ca(NO3)2, l00 μM NaFeEDTA, 30 μM H3BO3, l0 μM MnCl2, l μM CuCl2, 15 μM ZnSO4, 0.1 μM (NH4)6Mo7O24, and 50 μM KCl. Pi-deficient medium was made by replacing 1mM KH2PO4 by 1mM KCl. Zn-free medium was made by removing the only source of Zn (ZnSO4). Seeds were sown on the plates and stratified at 4 °C in the dark for 3 d. Plates were then transferred in a growth chamber for 20 d, day 1 of growth being defined as the first day of exposure of stratified seeds to light. Plants were grown under long-day conditions .The Pi measurements were performed as described by UBQ10: At4g05320) and expressed as relative values against wild-type plants grown in complete (+Pi and + Zn) medium. Quantification of the relative transcript levels was performed using the comparative CT method  and RQ1 RNAse-free DNAse (Promega) were used to prepare total RNA free of residual genomic DNA from 100mg frozen tissue, with shoot and root being analysed separately. Total RNA was quantified with a NanoDrop spectrophotometer (Thermo Scientific). cDNA was synthesized from 2 μg total RNA using an oligo(dT) primer and M-MLV reverse transcriptase (Promega). Real-time quantitative reverse-transcription PCR (qPCR) was performed with a LightCycler 480 Real-Time PCR System using SYBR green dye technology (Roche). PCR reactions (10 μl) containing 500nM each forward and reverse primers, 5 μl SYBR Green I Master, and 3 μl of a 1:3 cDNA dilution. Reactions were performed in LightCycler 480 Multiwell Plates 384 (Roche) covered with optical film (Sarstedt). A list of primers, efficiency of amplification, and specificity of the amplified PCR products have been described by g and 4 °C for 10min. The GUS enzymic assay was started by mixing 100 μl GUS assay buffer  NaN3, and 2mM 4-methylumberlliferyl-β-d-GlcA) with 20 μl protein extract in a 96-well microtitre plate, and was incubated at 37 °C. The amount of fluorescent product 4-methylumberlliferyl (4-MU) produced in the reaction was measured over time using a Fluoroskan-II luminescence spectrophotometer. The GUS activity was calculated as pmol 4-MU formed per μg total protein per h.GUS staining was performed on transgenic plants grown in agar-solidified media. Stained tissues were conserved in 70% ethanol at 4 °C after a progressive dehydration obtained from incubation in a graded series of ethanol, followed by embedding in LR White medium-grade resin or paraffin  leaves. Localization of PHO1;H3-GFP in Arabidopsis root and its colocalization with various marker proteins in transiently transformed tobacco epidermal cells was performed using a Zeiss LSM 700 confocal microscope with an Apochromat ×63 water immersion DIC objective with a 1.2 NA. Binary vectors with cDNA of the Golgi marker ManI-RFP (AT1G51590) (trans-Golgi marker SYP61-RFP (AT1G28490)  and using the Tukey’s test to compare mean values.Arabidopsis wild-type plants and pho2 mutant plants but not in phr1 and pho1 mutant plants. In order to determine the effect of Zn deficiency on Pi distribution in Arabidopsis tissues, plants were grown in media with either low Zn (0 μM Zn) or low Pi (10 μM Pi) and compared to those grown on complete medium (1mM Pi and 15 μM Zn). Mutants affected in interorgan Pi distribution or Pi signalling were included in this experiment, namely pho1, pho2, and phr1. The pho1 and pho2 mutants are characterized by accumulation of low Pi and high Pi in shoots, respectively  was observed. Overall, these results imply the existence of a process leading to the accumulation of Pi in the shoots of plants under Zn deprivation. PHO1 and PHR1 participate in this process.Zn deficiency increases the Pi content in the shoots of ectively . The phred genes . As expent media . In rootArabidopsis, this work analysed the transcript abundance of several genes involved in this pathway, namely PHR1, miR399b, miR399d, PHO2, and two Pi-starvation-responsive genes that are dependent on PHR1, namely AT4 and IPS1 . With the exception of PHT1;1, which was controlled at the transcription level by both Pi and Zn status of the plant  gene under the control of the PHO1;H3 promoter  leaves that were coinfiltrated with various subcellular markers. PHO1;H3-GFP did not colocalize with either the plasma membrane marker CBL1-OFP or the endoplasmic reticulum marker ER-rk-mCherry (trans-Golgi (TGN) marker RFP-Syp61 . In order to analyse the role of PHO1;H3 in transport of Pi within the plant, this work measured Pi content of shoots and roots in wild-type and pho1;h3 mutant plants . Collectively, these results indicate that the regulation of Pi acquisition from the soil solution may differ between plants species in response to Zn deficiency. It is, however, interesting to point that unexpectedly the transcript abundance of PHT1;1 is upregulated in the shoots of Arabidopsis Zn-deficient plants. The hypothesized primary function of PHT1;1 is the uptake of Pi from the soil and apoplastic space, particularly in roots (The identification of key genes involved in the coregulation of micro- and macronutrient homeostasis has become a major focal interest. In this work, in addition to nditions . This fiPHO1;H3 as an important player in the crosstalk between Zn deficiency signalling and the regulation of Pi homeostasis in Arabidopsis. Indeed, PHO1;H3 was significantly induced by Zn deficiency (pho1;h3 mutant accumulated similar levels of Pi as the wild type in the control condition, but accumulated significantly more Pi than the wild type when grown in Zn-free medium (PHO1;H3 under the control of the PHO1 promoter failed to complement the pho1 mutant (PHO1;H3 negatively regulates PHO1 activity in Pi transport to the shoot under Zn deficiency. This hypothesis is supported by the observation that PHO1 appears to be necessary for the overaccumulation of Pi in the shoot in response to Zn deprivation and that the pho1 pho1;h3 double mutant displays an equivalent Pi content in the shoot as the pho1 mutant regardless of the availability of Zn in the culture medium (pho2 mutant is characterized by Pi overaccumulation in shoots, leading to Pi toxicity (Arabidopsis, it has already been shown for sulphate (This work identified ficiency . Furthere medium . This ree medium  and 7 coe medium . It is t


An association between leukoencephalopathy, a disease of the white matter of the brain, and smoking heroin is well recognized. This paper describes 27 cases of leukoencephalopathy identified in two cities in British Columbia, Canada 2001-2006; the largest number of geographically and temporally defined reported cases in North America.Twenty cases of leukoencephalopathy were identified in and around Vancouver with onset dates December 2001 to July 2003; seven further cases were identified in Victoria September 2005-August 2006. Twenty (74%) of all cases were male, two couples were reported and eleven cases (55%) had Asian ethnicity. One case reported smoking heroin on a single occasion and developed mild symptoms; all other cases were hospitalized. Thirteen (48%) cases died; all had smoked heroin for a minimum of 3 years. Testing of one available heroin sample identified no substance other than common cutting agents.Although a specific etiology was not identified our study supports the theory of an intermittent exposure to a toxic agent added to the heroin or a combustion by-product. It also suggests a dose response effect rather than genetic predisposition. Collaboration with public health, health professionals, law enforcement and persons who use illegal drugs, will facilitate the early identification of cases to enable timely and complete follow-up including obtaining samples. Testing of implicated heroin samples may allow identification of the contaminant and therefore prevent further cases. It is therefore important to ensure key stakeholders are aware of our findings. Leukoencephalopathy refers to disease of the white matter of the brain and therefore can involve motor, sensory, and visual systems. Leukoencephalopathy can also disrupt cognitive and emotional function. There are many etiologies of leukoencephalopathy, including genetic disorders, cerebrovascular disease, eclampsia and toxic exposures. The clinical manifestation of the disease is a reflection of the areas of the brain involved in the disease process. Clinical features range from inattention, forgetfulness and personality changes, to dysarthria, ataxia, dementia, coma and death . TolueneAn association between leukoencephalopathy and smoking heroin has been recognized for over 25 years, although the exact pathogenesis is still not well understood. In 'chasing the dragon', heroin is placed on a piece of aluminium foil, heated with a flame from below, and the resulting vapour (pyrolysate) is inhaled with a straw or other tube-like structure. The practice was first recognized in Hong Kong in the 1950's but has now spread to users worldwide .Although the practice of 'chasing the dragon' is not uncommon, the associated leukoencephalopathy has been rarely reported. The first and largest outbreak of leukoencephalopathy linked to chasing the dragon was reported 1982 in the Netherlands, and included 47 cases, 11 23%) of whom died . Since 1% of whomBetween December 2001 and July 2003, 20 cases of leukoencephalopathy linked to heroin inhalation were identified in and around Vancouver, British Columbia (BC) Canada. An investigation was initiated at the time; but no causative agent was identified. Case reports were created to describe the clinical, pathologic, and imaging findings and awareness campaigns were initiated.In the fall of 2005, further cases of leukoencephalopathy were reported in Victoria, BC on Vancouver Island. An investigation was initiated in order to describe the epidemiology of the new cases. The purpose of this paper is to characterize all cases of heroin associated leukoencephalopathy identified since 2001 to date in BC in order to guide future research and public health actions.A case of heroin associated leukoencephalopathy was defined as:A person with clinical features of toxic leukoencephalopathy +/- neuroimaging with white matter changes typical of heroin-associated leukoencephalopathyAND a history of chasing the dragonAND a resident of BC or reported obtaining heroin in BCA case report form  and a fact sheet for physicians and the public were developed. The Provincial Health Officer notified BC neurologists about the cases of leukoencephalopathy associated with 'chasing the dragon' through the provincial specialty society list serve. Medical Health Officers throughout the province were also informed; they in turn notified the hospital Emergency Department heads and family physicians in their health regions. All physicians were requested to notify cases to local public health, which ensured the completed case report form was sent to the BC Centre for Disease Control.Where possible, charts of cases were reviewed and interviews were conducted with the case or next of kin. Abstracted information included: sex, date of birth, residence and ethnicity, medical, social and drug histories, date of symptom onset, date of hospital admission, clinical course and outcome. Cases were asked if a heroin sample was available for testing. A process was arranged for samples to be transported by the police to the Health Canada Drug Analysis Laboratory for content analysis using liquid chromatography-mass spectrometry.In addition to 20 cases identified in and around Vancouver with onset dates between December 2001 - July 2003, a further 7 cases were identified in Victoria with onset between September 2005- August 2006  cases. One case that reported smoking on a single occasion developed mild symptoms and was not hospitalized. Excluding this case the mean duration of chasing the dragon was 9.5 years (range 0.5 - 30 years). Of the cases that died, the minimum time of smoking heroin was three years. Use of other illicit drugs was reported in both Vancouver and Victoria cases . Three (43%) of the Victoria cases reported that smoking heroin was their only type of illicit drug use. At least six (86%) of the Victoria cases were taking methadone at the time of symptom onset; and five (25%) Vancouver cases reported using methadone, although these data are incomplete.Drug supply information was available for four (57%) of the Victoria cases; all reported obtaining heroin through telephone order and home delivery  from a male Asian supplier. The first three Vancouver cases also reported being supplied by an Asian supplier through dial-a-dope but data are incomplete for the rest of Vancouver cases.Three (47%) of the Victoria cases reported commercial painting as an occupation. No associations were found with type of aluminium foil ; or with other underlying conditions or medication other than methadone. No difference in colour, texture or smell of the heroin was reported by the cases. Apart from the two couples no cases reported knowing anyone else with similar symptoms. One heroin sample from a Vancouver case was tested with only common cutting agents identified.We have characterized 27 cases of leukoencephalopathy associated with 'chasing the dragon'. This is the largest number of cases reported in North America which are temporally and geographically defined. Although an etiologic agent has not been identified, we have a better understanding of the population at risk. The age, sex and ethnicity of our cases are consistent with the demographic profiles in other published reports. Forty-one percent of BC cases were Asian; this preponderance, also found in other studies, is likely representative of persons who 'chase the dragon' .Identification of two heterosexual case-couples suggests that the risk factors for leukoencephalopathy are more likely to be substance related rather than due to genetic predisposition. The distribution of the cases in place and time suggests a common intermittent exposure. Substances added to the heroin may be an inert 'cutting agent' such as caffeine, lactose or mannitol to increase the volume and hence profit, or an 'adulterant', which is added for its pharmacological effect . The diaAccording to a recent report from the UN Office of the Drug Commission, 96% of heroin seizures (2002-2007) in the US originated from Mexico and Columbia; whereas 98% of heroin seized in Canada originated from Southwest Asia . Althouget al found that chasing the dragon was a well-established method of using heroin in certain populations, not merely a pre-injection phase of heroin addiction [The purity of street heroin in BC, determined by Health Canada, Drug Analysis Service, has increased from 5-10% in the 1970's, to greater than 60% . Howeverddiction . TherefoOne case with a single brief exposure to inhaling heroin pyrolysate required outpatient support only. This finding was similar to a case report in the literature of a patient with an isolated exposure who had a complete recovery, and is consistent with a dose-response relationship ,12. ClarReported clusters of leukoencephalopathy have been associated with smoking heroin. However isolated cases of leukoencephalopathy associated with using heroin and heroin and cocaine intravenously -32, and The etiology of heroin-related toxic leukoencephalopathy requires further research and public health involvement. The severity of the outcome and lack of curative treatment highlights the importance of future investigations. Current therapy with coenzyme Q and vitamin supplements is anecdotal only . PreviouLimitations of our study include incomplete case and drug information. This may be related to the inability of cases to communicate at presentation and the rapid decline in mental state of some. Also, the illicit nature of drug use may cause concerns about sharing information regarding other drug users or the source of heroin. Family members may have little prior knowledge of the case's drug use, limiting the information provided through collateral history. The delay between heroin use and symptom onset also reduces the likelihood that implicated heroin is available for testing.Recognizing the difficulties inherent in studying such a sporadically occurring condition, a serious effort to determine etiology may require both reactive and prospective approaches. Collaboration with public health, health professionals, law enforcement and persons who use illegal drugs, would facilitate the early identification of cases to enable timely and complete follow-up including obtaining heroin samples. A pre-arranged process for transporting and testing implicated heroin samples may allow identification of the contaminant and therefore prevent further cases. Purity and contaminant sampling programs for street drugs could also be considered. It is therefore important to ensure key stakeholders are aware of these findings and the association of leukoencephalopathy and heroin smoking.The authors declare that they have no competing interests.JAB oversaw and gave input at all levels of the investigations. ML and LC followed up the cases in Vancouver. RS and LS followed up the cases in Victoria. LC wrote a report on Vancouver cases. RS wrote the first draft of the paper. NA performed an updated literature review and edited the paper. SDS provided clinical expertise. All authors have read, given input and approved the final manuscript.


Nerve injury increases expression of glutamate receptors and their associated Homer scaffolding proteins throughout the pain processing pathway. Homer proteins, and their associated glutamate receptors, regulate behavioral sensitivity to various addictive drugs. Thus, we investigated a potential role for Homers in the interactions between pain and drug reward in mice. Chronic constriction injury (CCI) of the sciatic nerve elevated Homer1b/c and/or Homer2a/b expression within all mesolimbic structures examined and for the most part, the Homer increases coincided with elevated mGluR5, GluN2A/B, and the activational state of various down-stream kinases. Behaviorally, CCI mice showed pain hypersensitivity and a conditioned place-aversion (CPA) at a low heroin dose that supported conditioned place-preference (CPP) in naïve controls. Null mutations of Homer1a, Homer1, and Homer2, as well as transgenic disruption of mGluR5-Homer interactions, either attenuated or completely blocked low-dose heroin CPP, and none of the CCI mutant strains exhibited heroin-induced CPA. However, heroin CPP did not depend upon full Homer1c expression within the nucleus accumbens (NAC), as CPP occurred in controls infused locally with small hairpin RNA-Homer1c, although intra-NAC and/or intrathecal cDNA-Homer1c, -Homer1a, and -Homer2b infusions (to best mimic CCI’s effects) were sufficient to blunt heroin CPP in uninjured mice. However, arguing against a simple role for CCI-induced increases in either spinal or NAC Homer expression for heroin CPA, cDNA infusion of our various cDNA constructs either did not affect  or attenuated (NAC) heroin CPA. Together, these data implicate increases in glutamate receptor/Homer/kinase activity within limbic structures, perhaps outside the NAC, as possibly critical for switching the incentive motivational properties of heroin following nerve injury, which has relevance for opioid psychopharmacology in individuals suffering from neuropathic pain.Pain alters opioid reinforcement, presumably  Comorbidity exists between chronic pain and motivational disturbances  mice , as well as several strains of constitutive gene knock-out (KO) mice that were available at the time of study, including The procedures for inducing peripheral neuropathy by CCI of the sciatic nerve were identical to those described recently by our group  over ice, homogenized in a buffer containing both protease and phosphatase inhibitors and subjected to conventional immunoblotting procedures (20 μg protein/lane) as described previously by our group  as published previously  were dissected from B6 mice  cDNA or the hemagglutinin (HA) tag fused to the coding region of rat n = 8–12/group). Immunoblotting data were analyzed by one-way analyses of variance (ANOVA) with Tukey’s multiple comparison post hoc tests and these results are presented in Table post hoc tests, when appropriate. To confirm significant place-conditioning, a priori dependent-sample t-tests were conducted for the time spent in the heroin-paired vs. -unpaired compartment, separately for each treatment group/genotype. α = 0.05 for all analyses and the results of the statistical analyses for the behavioral assays are presented in their corresponding figure legends.Behavioral and biochemical results are presented as means ± SEM n = 8–2/group. Chronic constriction injury of the sciatic nerve increased mechanical and cold hypersensitivity in B6 mice Figures A,B. Thist-tests, p > 0.05); (2) saline- or heroin-induced locomotor activity on injection 1 or 4; or (3) the expression of heroin-induced locomotor sensitization, which was observed only at the 3-mg/kg dose .We next assayed for CCI-induced changes in heroin-conditioned reward in B6 mice as an index of motivation. All but the lowest heroin dose elicited a significant CPP in injury-naïve B6 controls Figure . In contHomer1a, Homer1, and Homer2 null mutant mice, as well as in transgenic mice with a disrupted mGluR5-Homer interaction (R/RGrm5). The 0.1-mg/kg heroin dose elicited a significant CPP in injury-naïve WT mice from all strains and this CPP was absent in all homozygous mutant littermate animals  heroin consistently supported CPP in injury-naïve WT mice. Remarkably, this low-dose heroin CPP was attenuated or absent in injury-naïve mice from all four mutant strains. Opioids and their withdrawal alter Homer1 deletion abolished low-dose heroin CPP, while intra-NAC shRNA-Homer1c infusion had absolutely no effect. These data indicate either that: (1) the neural locus mediating the CPP effect of Homer1 deletion resides outside the NAC or (2) the CPP effect of Homer1 deletion reflect an absence of inducible, rather than constitutively expressed, Homer1 gene products. As the effects of Homer1a deletion mirrored those of Homer1 deletion argues in favor of the latter possibility. However, based on suggestions of regional differences in heroin-induced changes in Homer1 mRNA within PFC, AMY, and dorsal striatum (Kuntz et al., Homer1 gene products in these other addiction-relevant brain regions may contribute more so to the conditioned incentive motivational properties of low-dose heroin. It is interesting to note, however, that intra-NAC cDNA-Homer1c, as well as intrathecal cDNA-Homer1a, -Homer1c, and -Homer2b infusion, in injury-naïve mice was sufficient to block heroin CPP. The result for the NAC may be counterintuitive based on the findings from the KO studies, but, as argued below, may reflect a facilitation of low-dose heroin hyperalgesia that renders the heroin experience more aversive.Interestingly, Most notable and distinct from the results of earlier CPP studies in injured animals (c.f., Niikura et al., Homer1 and Homer2 KO mice exhibited conditioned approach behavior following nerve injury. That both Homer1a deletion and the R/RGrm5 transgene exacerbate neuropathic pain symptoms, while neither Homer1 nor Homer2 deletion influence pain hypersensitivity (Obara et al., Indeed, the present data from the studies of transgenic mice support this possibility as no evidence for CCI-induced heroin CPA was apparent in any mutant strain; in fact, both The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


MET/AKT cascade affects neurodevelopment in general populations and face recognition in patients with autism. This study explores the possible role of MET/AKT cascade in facial emotion perception.Facial emotion perception is a major social skill, but its molecular signal pathway remains unclear. The MET  and AKT rs1130233 were genotyped and tested for their effects on facial emotion perception. Facial emotion perception was assessed by the face task of Mayer-Salovey-Caruso Emotional Intelligence Test (MSCEIT). Thorough neurocognitive functions were also assessed.One hundred and eighty two unrelated healthy volunteers (82 men and 100 women) were recruited. Four single nucleotide polymorphisms (SNP) of MET rs2237717, individuals with the CT genotype performed better in facial emotion perception than those with TT , and showed no difference with those with CC. Carriers with the most common MET CGA haplotype (frequency = 50.5%) performed better than non-carriers of CGA in facial emotion perception . In MET rs2237717/AKT rs1130233 interaction, the C carrier/G carrier group showed better facial emotion perception than those with the TT/AA genotype , even when neurocognitive functions were controlled (p = 0.046 by GLM).Regarding MET variances and MET/AKT interaction may affect facial emotion perception, implicating that the MET/AKT cascade plays a significant role in facial emotion perception. Further replication studies are needed.To our knowledge, this is the first study to suggest that genetic factors can affect performance of facial emotion perception. The findings indicate that  Facial emotion perception is a key element of social function Several studies, focusing on monoamine pathways, suggest that genetic variances can determine activities of relevant brain regions when people perceive facial emotion. Non-depressed adults with short allele of deletion polymorphism in the serotonin transporter-linked polymorphic region (5-HTTLPR) have impaired emotion processing and difficulty disengaging attention from facial stimuli However, whether genetic factors can affect performance of facial emotion perception remains uncertain. In addition, the signal cascades of facial emotion perception deserve further studies because accumulating evidence suggests that regardless of the underlying genetic complexity, the pathophysiology and endophenotypes of schizophrenia may be determined by a small number of common signaling pathways MET, located at chromosome 7q31, is expressed in amygdala, hippocampus, and cerebral cortex MET is vital for cerebral and cerebellar development MET is associated with autism MET promoter SNP rs1858830 has been shown to alter gene transcription MET protein MET activates phosphorylation of AKT, and this cascade is essential for anti-apoptotic signaling AKT gene is also associated with schizophrenia AKT may be involved in the dopamine neurotransmission pathway AKT can affect amount of AKT protein in lymphocytes and postmortem frontal cortex and hippocampus regions AKT rs1130233 have impaired executive function, processing speed, trail making, verbal and category fluency and prefrontal cortical structure AKT in emotion perception requires elucidation.MET and AKT cascade on facial emotion perception in healthy individuals.This study aimed to testify the genetic effects of the This study was approved by the Institutional Review Board of China Medical University Hospital (CMUH), the authors' institutional review board, and performed in accordance with the Declaration of Helsinki. After complete description, all subjects gave written informed consent.The participants were 182 unrelated Han Chinese healthy individuals (82 men and 100 women) with a mean age of 31.52  years and a mean education level of 14.91 . All were Han Chinese living in Taiwan. They were free of any axis I or II psychiatric disorders, as determined by a research psychiatrist using the Structured Clinical Interview for DSM-IV This study utilized the face task of the emotion perception branch of Mayer-Salovey-Caruso Emotional Intelligence Test (MSCEIT) An experienced research psychologist comprehensively assessed cognitive functions to control for their possible effects when we explored genetic effects on facial emotion perception. Among all 182 subjects, 149 also received thorough cognition assessments with a battery of tests, which were the same as or the analogues of tests from Measurement and Treatment Research to Improve Cognition in Schizophrenia (MATRICS) Peripheral bloods from schizophrenia subjects were collected in EDTA-tubes. The DNA was isolated by employing the salting-out method MET gene and rs1130233 of the AKT gene were selected due to associations with schizophrenia Four SNPs  of the MET gene were: for rs2237717, sense primer 5′- CCA CGT ACT TCA TCA ATG -3′ and antisense primer 5′- CTT CCT GGC AAT AAA GAG -3′; for rs41735, sense primer 5′- GCT ATT GGA AAA GAA AAG GAT AGA AAC -3′ and antisense primer 5′- CCA TCT GTA GTT GGT AGA ATA TCT CT -3′; for rs42336, sense primer 5′- AGA GAA CTA GAT TAC GTC AGC CAA AGA -3′ and antisense primer 5′- TCT GCC CTG GGG TCA CAT -3′. For rs1130233 of AKT, sense primer 5′-GCT GTT CTT CCA CCT GTC -3′ and antisense primer 5′-AGG GCT GAC ACA ATC TCA -3′. Neither a TaqMan Assay-by-Design nor a high resolution melting method (HRM) in ABI 7500 Fast Real-time PCR system (Life technologies) was able to reliably provide rs1858830 genotype from genomic DNA, probably because of ≈85% GC in the region. Therefore the genotype at rs1858830 was determined by direct resequencing.The sequences of the primers for the 2 concentration. The cycling conditions were as follows: denaturation at 95°C for 4 min, followed by 35 cycles of 95°C for 30 s, 58–64°C for 30 s , and 72°C for 45 s, followed by one HRM cycle of 95°C for 10 s, 60°C for 1 min, and continuous acquisition to 95°C for 15 s (ramp rate 1%), and subsequently down to 60°C for 15 s. The HRM data were analyzed using the high resolution melting (HRM) software, version 2.0.1. All melting curves deviating from the wild-type curve and appearing as a different color in discrete plots contain a variant potentially. In addition, 24 randomly selected samples including three genotypes of one SNP which identified by HRM were sent to direct sequencing by Mission Biotech CO., LTD, Taiwan.PCR was performed in a 20-µl volume containing the following: 1× PCR reaction buffer, 1 U FastStart Taq DNA Polymerase (Roche), 0.3 µM of each primer, 1.5 µM SYTO® 9 green fluorescent nucleic acid stain (Life technologies), and 20 ng DNA template, with a 2 mM final MgClFigure S1). MET haplotype and diplotype were reconstructed via SAS/GENETICS by implementing the PROC HAPLOTYPE procedure (SAS® 9.1 software). Because MET could induce phosphorylation of AKTMET and AKT may be present in facial emotion perception. Therefore, this study divided subjects into four groups according to the genotype status and function assessment in the previous studies MET (rs2237717) and AKT (rs1130233) genotypes. The four groups of MET/AKT were TT/AA, TT/G carrier, C carrier/AA, and C carrier/G carrier.All the statistical analyses were performed with the Statistical Package for the Social Sciences (SPSS), version 17.0 for Windows. Deviation of the genotype counts from the Hardy–Weinberg equilibrium was tested by employing a Chi-Square goodness-of-fit test. Chi-Square test was used for gender comparisons between genetic groups. Age, years of education, facial emotion perception and composite cognition between genetic groups was test by ANOVA. Bonferroni correction was used in post-hoc analysis. Linkage disequilibrium structure was examined using Haploview 4.2 MET, AKT, and MET/AKT combination on facial emotion perception, and control variables were age, gender, and years of education as covariates or fixed factors as appropriate. This study utilized partial η2 (eta squared) by SPSS d) MET, AKT, and MET/AKT status to testify whether the combined gene effect was additive or synergistic. Finally, this study utilized G* power 3 p-values were below 0.05 (two-sided).General linear models (GLM) were utilized to test the main effects of MET and AKT rs1130233 did not deviate from the Hardy-Weinberg equilibrium. Genotype and allele frequencies of the five SNPs were similar to those of other studies Table S1). There were high linkage among MET rs2237717, rs41735, and rs42336 (D′>0.9); therefore, we constructed haplotype block among the 3 SNPs . In this haplotype block, the most common two haplotypes from MET rs2237717, rs41735, and rs42336 were CGA and TAG . There were only 2 subjects with CAG haplotype, and their mean [SD] facial emotion perception (88.9[11.0]) was similar to that (84.3[20.5]) of the TAG group; we therefore combined CAG with TAG as the non-CGA group.All four SNPs of MET genotype, AKT genotype, MET haplotypes, and MET-AKT are shown in MET genotypes, AKT genotypes, or CGA haplotype.Demographic characteristics and emotion perception performance by MET rs2237717 . Bonferroni post hoc analysis revealed this difference was driven from difference between CT and TT . There was a dominant effect of the C allele of MET rs2237717, since there was no difference between CT and CC in terms of performance. No differences of facial emotion perception were observed in the other three MET SNPs and AKT rs113023 . Subjects with MET CGA haplotype also performed better than non-CGA individuals in facial emotion perception by GLM analysis (p = 0.009) . The Bonferroni post-hoc analysis showed that the difference was driven from the comparison between the individuals who were simultaneously C carriers and G carriers and those with TT/AA . In addition, there was nominal difference between TT/AA and C carrier/AA (p = 0.049); however it did not survive after Bonferroni correction (p = 0.296).Considering the combined effect of MET rs2237717increased facial emotion perception by 9.987, 8.766, respectively  when compared with TT genotype. As for MET rs42336, AA homozygotes had better performance than those with GG genotype by 8.686 (p = 0.041). Subjects with MET CGA haplotype had better emotion perception by 8.748 than those without this haplotype (p = 0.009). However, MET rs41735, rs1858830 and AKT rs1130233 did not affect facial emotion perception (p = 0.048) in general linear regression analysis for MET rs2237717. Other confounding variables did not significantly influence facial emotion perception.As shown on rception Table 2.MET and AKT variants, we chose MET rs2237717 and AKT rs1130233, because MET rs2237717 had the most significant influence on facial emotion perception in the current study and AKT rs1130233 had been shown to be functional in the previous studies MET/AKT on facial emotion perception was significant (p = 0.041). When the individuals who were C carrier and G carrier simultaneously were used as the reference group, their facial emotion perception was better than that of those with TT/AA  were similar to those in the all 182 subjects. MET rs2237717, MET CGA haplotype and MET/AKT combination still had significant effects on facial emotion perception (Table S2). MET and AKT variants had no significant influence on cognitive function (Table S2). Effects of MET/AKT variants were also analyzed by general linear regression analysis in the 149 subjects. After control for age, gender, education duration, and neurocognition, subjects with CC and CT genotypes of MET rs2237717 had better facial emotion perception when compared with TT genotype. Subjects with MET CGA haplotype had better emotion perception than those without this haplotype (p = 0.020) (details not shown). The participants who were C carrier/G carrier of MET/AKT were superior to those with TT/AA in facial emotion perception . The possible reasons include differences in cognitive tests, ethnicity, and haplotypes between studies.Burdick et al AKT rs1130233 was reported to be associated with reduced cognitive functions Table S1) (perhaps due to the modest sample size). AKT is also involved with emotional memory learning in the amygdala, which is a critical area in emotion perception AKT variants on facial emotion perception, the present study revealed that AKT had additive effect with MET on facial emotion perception: the individuals who were both C carriers/G carriers of MET/AKT had better emotion perception than those with TT/AA. Therefore, this finding suggests that the AKT gene may modulate the MET effect on facial emotion perception. Such gene-gene interactions are pretty common in CNS signal pathways. One recent example is the interaction of Neuregulin-1 (NRG-1) and its receptor, ERBB4 NRG1/ERBB4/AKT risk genotypes were disproportionately worse in dorsolateral prefrontal function in the image study The A allele of MET genotype, haplotype and MET/AKT combination still exerted significant effects on facial emotion perception after control for education level.Consistent with a previous study MET and MET/AKT was medium to large. Future studies with larger samples in other ethnicities are warranted.The present study had some limitations, such as utilizing the face task of the emotion perception branch of MSCEIT, which only reflects the general ability of facial emotion perception. However, the face task of emotion perception can also be an important component of social cognition MET/AKT cascade may play a role in facial emotion perception. Further studies in other races or in patients with mental disorders such as schizophrenia are needed.In summary, the results suggest that the Figure S1MET SNPs was computed using Haploview 4.2.Linkage disequilibrium (D′) for the (TIF)Click here for additional data file.Table S1MET SNPs and AKT SNP.Allele frequencies of (DOC)Click here for additional data file.Table S2MET SNPs and haplotypes, AKT SNP, and MET/AKT variants in the 149 patients who also received thorough cognitive assessments.Demographics, facial emotion perception, composite cognition*, and (DOC)Click here for additional data file.


Human Chagas disease originated in Latin America, being spread around the world in relation with multiple bioecological, sociocultural, and political factors. The process of the disease production and dispersion is discussed, emphasizing the human migration and correlated aspects, in the context of globalization. Positive and negative consequences concern the future of this trypanosomiasis, mainly in terms of the ecologic and sociopolitical characteristics of the endemic and nonendemic countries. Trypanosoma (Schizotrypanum) cruzi (T. cruzi), HCD progressively became engaged with the political context of endemic countries and with a low standard of living of their population [In a broad sense, human Chagas Disease (HCD) reflects the bioecological, historic, and social situations of Latin America (LA), presenting a remarkable medical and social impact in the region , 2. In ipulation –5. Follopulation –9. In thpulation , 10. In Until the last century, HCD affected primordially rural people living in poor dwellings colonized by the vector insect. It has been extremely impacting disease, with expressive mortality among children in its acute phase and severe heart lesions in about 10–20% of chronic adult patients. Its medical and social burdens involve mortality, high hospital and social costs, absenteeism, and labor incapacity –13. For Social and political changes of the whole society have been very expressive in the last decades, following the process of globalization, all around the world. Among other factors such as the increasing of human migration, globalization has produced an economical effect tremendously unequal among different countries and social groups, as a consequence of economical speculations, international and supranational competition and political affairs , 26.pud Dias (2007)). Neoliberalism, a main axis of globalization, represents a movement to benefit the great world potencies and the multinational enterprises. Poor countries usually suffer with neoliberal policies, in terms of underemployment, low salaries, inequity, and strong dependency of international capitals and of the global market . Particularly, this situation used to be responsible for the intensive migration process all around the world. Regarding LA, after two decades of Neoliberalism, the World Bank evaluation in 2005 considered that the results (in terms of economic grow) remained much lower than it was formerly awaited [Regarding the overcoming of neglected diseases, Dr. Margareth Chan, Director-General of WHO, said very clearly that “efforts to control neglected tropical diseases constitutes a pro-poor strategy on a grand scale. The logic has changed: instead of waiting for these diseases to gradually disappear as countries develop and living conditions improve, a deliberate effort to make them disappear is now viewed as a route to poverty alleviation that can itself spur socioeconomic development” . In othe awaited , 26.In the poor endemic regions of LA, the impact of globalization has been considerable in terms of HCD epidemiology, management, and prevention , 28. Thechagasic population usually has no political density, being unable to obtain the minimum social gains [Regarding the social and political evolution of LA, HCD can be considered a reflex of the regional history, particularly in terms of equity and production dealings. By the side of the infected individuals, globalization and market implications produced bad and good results, spreading the disease and improving the conditions for medical access. In general, the market related with HCD is very weak; the poor chagasic individuals usually depend on public health, especially in poor endemic countries. The main commercial profits (relatively low) concern laboratory diagnostic reagents and insecticides . In addial gains , 24, 28.al gains , 32. In al gains , 24. On al gains , 14, 29;al gains , 16, 33.al gains , Dias [2al gains , and Schal gains  observe It can be said that in the beginning, the spreading of HCD in Latin America was related to human movements, in parallel with progressive rural settlements and the domiciliation of triatomines , 8, 29. In Rio de Janeiro city, amongst 260 infected individuals, Coura verified that all of them originated from endemic rural zones of Brazil , Bolivia, and Paraguay . In ArgeDeparting from the basic rural situation, different flows of individuals and families can be detected along the history of LA: (a) migration inside different rural areas, resulting in vector and reservoir dispersion; (b) migration from endemic to nonendemic rural areas (it is the case of thousands of families in Brazil going to Amazon region); (c) migration from rural localities to urban centres, generally being installed in peripheral sites; (d) the reverse migration from urban to rural areas; (e) the seasonal migration of rural laborers who travel periodically to work in different unskilled labor fronts such as sugar cane crops, petrol exploration, and civil constructions; and (f) migration from endemic to nonendemic countries, departing both from rural and urban situations. Basically, HCD and human migration are correlated with the social process of poor individuals moving themselves to look for better conditions of life , 13, 47.Sosa  analyzedAnalyzing rural emigration in endemic areas, several authors agree that its basic cause is a complex process involving the failure of the classic familial agriculture, the boom of the urban-industrial model, and the new agroindustrial perspectives for massive production. In addition, the lack of a social policy concerning agriculture in Latin America in the last century also stimulated the rural emigration: no prices, no technical assistance, and no proportion between efforts and results , 29, 30.The general situation concerns the migration of infected people from Latin America, particularly since the end of the XIX century. Many authors have been dedicated to this subject, the consensus being that the basic cause involves a survival strategy of poor population in search of employment and better living conditions in more developed countries , 50–52. T. rubrofasciata and Linshcosteus sp. in Southern Asia [Several considerations have been made about this important theme, involving complex social and epidemiological aspects , 19, 50.ern Asia , 39, 51.ern Asia , 50, 52.T. cruzi dispersion. Climatic and other ecologic factors, but mainly anthropic actions, are naturally involved in such movements. Regarding triatomine vectors, different ecotopes and domiciliation (colonization of human dwellings) depend chiefly on the species and ecologic situations [Triatoma infestans to their stone and mud made houses. Once colonized, the species became more and more adapted to human houses, being able to produce enormous indoors colonies. From the first focus to neighbour houses, the vector is able to disperse by means of active or passive migration. From Bolivia, T. infestans reached Chile, Peru, Argentina, Uruguay, and Paraguay, mainly at the expense of human displacements due to agriculture expansion and internal wars [Other different situations must be focused on, not only those related to people displacement. Vectors and reservoirs are also in constant movement, being implied in the whole process of tuations , 36, 53.tuations . Originatuations , 35. Thenal wars , 38.T. infestans spreading in Brazil since the XIX Century is emblematic, well studied by Silva [T. infestans, originated from Paraguay and Argentina (Jesuit Missions) and from Uruguay (regular commerce). The troops carried passively the triatomines to S. Paulo during more than 80 years, chiefly arriving in the Sorocaba region, which became the epicenter of T. infestans dispersion in S. Paulo. The process of infestation was progressive and continuous, in direction to the west of the state, as well as to the states of Paraná and Minas Gerais, following the expansion of the coffee frontier. Coffee, the so-called green gold, became in São Paulo the basis of a new economic era, impelling immigration, deforestation, urbanization, and industrialization [The history of by Silva . The explization , 38. Latlization .Triatoma dimidiata being carried passively from wild ecotopes of Costa Rica to human urban and peri urban dwellings by means of firewood transportation, a process that was being reduced when the population changed fire wood cooking to petrol gas and electricity [Another example refers to ctricity .R. prolixus seems to have a very complex story of its spreading from Venezuela to Mexico and Central America, involving an international scientific interchange of insect collection , the sea commerce between Central and South America, and even, possibly, the dispersion of eggs and nymphs, carried passively in birds migration [Passive migration and active displacements of the vector are present in several other reports, including at the international level. For instance, igration . Triatomigration , 55. Theigration , 51, 54.Natural reservoirs. In parallel with vectors movement, mammalian reservoirs and their mobilization in the vicinity of human houses play an important role in HCD epidemiology, particularly the so-called synanthropic reservoirs such as the opossums [T. cruzi strains, wild reservoirs can introduce the parasite in domestic cycle, when they get closer to human dwellings harboured by triatomines. Such reservoir movements many times are a natural consequence of human intervention in natural ecotopes, chiefly when their natural shelters and food sources are destroyed [opossums , 10, 14.estroyed , 14, 53.estroyed , 36.T. cruzi also has some implications for globalization. Human intensive movements and progressive modifications of sylvatic ambient  are clearly changing the general landscape since America discovery. Particularly the emergence of several acute cases of oral transmitted HCD has been important, especially in Amazon region, basically dependent on the contamination of a series of meals of T. cruzi originated from wild triatomines [T. infestans and R. prolixus, besides a residual peridomestic infestation by ubiquist species . By another way, due to anthropic affairs, wild species such as P. geniculatus, R. pictipes, T. rubrofasciata, and T. picturata could occasionally invade human dwellings, eventually establishing little colonies and being able to transmit HCD [T. cruzi than with its exacerbation [deforestation resulting from a strong wood market and the expansion of agroindustries in endemic areas;the extensive use of pesticides in agroindustrial projects;the expansion of the use of electricity and industrial machinery, interfering with wild triatomine behavior and influencing the rural demography;the progressive reduction of mammal reservoirs of the parasite, resulting from deforestation, pesticides, and extensive monocultures;progressive changes in the productive model, reinforcing capitalist agroindustries in detriment of the classical strategy of family subsistence; in the same logic, the dominant market and large scale economy will overlap the classical microeconomies; the modernization of agriculture, specially by means of automatic tools, robotics, and housing improvement, expulsing poor familial economies and hampering triatomine domestic colonization.As a consequence of urbanization and control programs, the wild cycles of the parasite play a particular role in the maintenance of HCD. It is expectable that with the decreasing of domestic cycles in endemic areas, the major risks of HCD incidence will depend on sylvatic triatomines and wild trypanosome populations in the vicinity of susceptible human beings , 53. Theatomines , 33, 49.atomines , 53, 54.smit HCD , 38, 55.erbation , 29, 56.erbation , 30, 50 the improvement of medical expertise for HCD management in chronic cases all over the world, mainly in terms of the primary health-care level; in corollary, the improvement of drugs and other medical proceedings is highly desirable, considering the elder patients and the superposition of other chronic and degenerative diseases;the betterment of medical and social security systems in order to assure adequate access and coverage for all infected individuals;ensuring political and administrative conditions to maintain at least two or three decades more the medical expertise able to manage adequately the infected individuals.Presently, all over the world, the increasing life expectancy has been a great tendency of the population, resulting from better medical and social assistance. The medical management of HCD requires new knowledge and practices in terms of disease physiopathology and of the superposition of several other medical problems occurring chiefly in higher age groups, such as hypertension, diabetes, coronary diseases, Parkinsonism and physiological denervation. In such a scenery, the medical management of HCD involves three major challenges, highly depending on the political and social organization all around the world , 52, 57:Other predictable situations could be emphasized for HCD management at the medium term, all of them being correlated with globalization. First of all, globalization has been a strong stimulus for the advance of medical security enterprises, able to increase the access of a progressively higher number of infected people to medical attention. The specific treatment is another important question, because it has been more and more indicated for chronic patients, trying to minimize and/or to prevent severe clinical conditions, specially advanced heart disease and sudden death. The basic problems considering this subject lie in diagnosis access, medical expertise, and drug availability, besides a good treatment adherence , 20, 57.chagasic individuals will continue to be poor, illiterate, and socially excluded. National and international migration certainly will continue, spreading infected individuals in urban spaces all over the world during two or three more decades. The overcoming of HCD will depend on the public sectors  and on the reduction of inequity. As a correlated issue, considering developed nonendemic countries such as Canada, the social situation of poor immigrated individuals usually involves personal constraints and severe consequents for their health, in terms of disease management and work safety [It is admitted that HCD and other so-called neglected diseases have received and will continue to receive strong influences of the globalization process. Poor, isolated, and marginal areas in LA will continue to exist, as remaining foci of disease prevalence and active transmission. Likewise, the majority of the k safety . Coming k safety .In terms of political strategy, macropolicies and program organization are points to remain in the agenda of WHO and of the governments of endemic countries by the next two or three decades, keeping alive the interest and the priority of HCD and its control. National and regional programs must be adapted to the decentralization of health systems, another universal consequence of globalization , 30. NewChagas Disease has been a concrete and impacting social and medical problem in LA, with multiple aspects associated with social iniquity and globalization. Human migration has been highly dependent on globalization and other social processes, being responsible for disease expansion and for a new epidemiologic situation, in which medical care must be improved. In spite of different financial and political constraints, HCD has been controlled, with remaining two or three more decades of program consolidation and medical attention for all infected people. This is a particular task for LA, because the fight against HCD requires the action of the state, as the basic social provider for the poorest citizens. The main strategies to face HCD have showed to be considerably effective at the medium and long terms, depending on social improvement, transmission control, and medical attention. Considering these points, the persistence of iniquity and other negative aspects of globalization have been hard challenges to be overcome in endemic areas. With the progressive reduction of its transmission and morbidity in the last two decades, the visibility of HCD as well as its political priority tends to decrease. In addition, the emergence of other public health problems such as dengue fever, influenza, and epidemic AIDS is contributing to the deviation of human and financial resources from the existing HCD programs , 16, 24.Also, the transition of health sector all over LA has been slow and complicated, in spite of its highly logical and stimulant theoretical approach. Decentralization of health services and the reduction of vertical programs have been a consequence of globalization, putting the major responsibility of medical care and control programs on peripheral governmental levels , 39, 51.


Post-translational regulation plays an important role in cellular metabolism. Earlier studies showed that the activity of plastidial starch phosphorylase (Pho1) may be regulated by proteolytic modification. During the purification of Pho1 from sweet potato roots, we observed an unknown high molecular weight complex (HX) showing Pho1 activity. The two-dimensional gel electrophoresis, mass spectrometry, and reverse immunoprecipitation analyses showed that HX is composed of Pho1 and the 20S proteasome. Incubating sweet potato roots at 45°C triggers a stepwise degradation of Pho1; however, the degradation process can be partially inhibited by specific proteasome inhibitor MG132. The proteolytically modified Pho1 displays a lower binding affinity toward glucose 1-phosphate and a reduced starch-synthesizing activity. This study suggests that the 20S proteasome interacts with Pho1 and is involved in the regulation of the catalytic activity of Pho1 in sweet potato roots under heat stress conditions. Starch is the main storage polysaccharide in plants. Several essential enzymes are involved in starch biosynthesis, including ADP-glucose pyrophosphorylase, starch synthase, branching enzyme, and debranching enzyme Pho1 gene of oryza sativa significantly affected the starch content and the size of mature seeds at 20°C, indicating that Pho1 may be required for normal starch biosynthesis in rice endosperm at low temperatures. However, an Arabidopsis thaliana mutant deficient in the Atphs1 gene, a homolog of Pho1, showed no evident change in starch biosynthesis or degradation, but was more sensitive to abiotic stress. These observations suggest that Atphs1 may be required for stress tolerance, and imply that Pho1 plays a vital role under certain environmental conditions Previous studies showed that the accumulation of starch is proportional to the expression and activity of Pho1 in potato tubers in vitro, and that the phosphorylated Pho1 showed higher sensitivity to proteolytic modification Due to the significance of Pho1 in higher plants, some attention has been given to the regulation of its activity. Most of the Pho1 isolated from mature potato tubers The ubiquitin-proteasome system is a tightly regulated pathway that controls protein levels in eukaryotes In this study we present evidence that the 20S proteasome associates with Pho1 and is specifically involved in the degradation of the L78 insertion in sweet potato roots. Our data also demonstrated that incubating sweet potato roots at 45°C accelerated the MG132 inhibited degradation of the L78 insertion by the 20S proteasome. Furthermore, the kinetic properties of Pho1 were altered after heat treatment, resulting in the proteolytic modification of Pho1. This study reveals that the 20S proteasome is involved in the regulation of Pho1 catalytic activity.During the purification of Pho1 from sweet potato roots by size-exclusion chromatography , we founTo further confirm the interaction between Pho1 and the 20S proteasome, a Pho1 specific monoclonal antibody, H7c, was used to immunoprecipitate Pho1 from the crude extract of sweet potato roots. The immunoprecipitant was separated by SDS-PAGE , and anaTo examine the components of HX in native and denatured conditions, a modified two-dimensional gel electrophoresis was applied to the experiment. HX was separated by native PAGE for the first dimension 1-DE) and then subsequently applied to the second-dimension SDS-PAGE 2-DE). The 2-DE gel stained with Coomassie Brilliant Blue R-250 (Coomassie) reveals that HX contains P110, F50s  and mult-DE. The -DE and td) revealed as multiple bands on the native PAGE still displays the catalytic activity in the direction of starch synthesis within 6 h of incubation  from heat-treated sweet potato roots  than Pho1d (Km  =  1.4 mM) (Kcat/Km  =  20.86 mM−1 s−1) than Pho1d (9.83 mM−1 s−1) in the direction of starch synthesis  . Consequynthesis .A canonical process for proteasome-mediated proteolytic modification of cellular proteins includes ubiquitination and the 19S proteasome activator Pho1 undergoes proteolytic modification in mature potato tubers in vitro experiment.In the present study, an unusual high temperature at 45°C and a long incubation time for up to 12 hours were applied to examine the degradation of Pho1 proteins . In the in vitroAlthough we found that Pho1 can be degraded by the 20S proteasome under a stringent heat stress condition, the signaling mechanism which initiates the degradation progress remains unclear. Phosphorylation was implicated as part of a regulation in targeting protein for ubiquitination and proteasomal degradation. I kappa B alpha is phosphorylated in response to various signals at two specific N-terminal Ser-32 and Ser-36; this leads to its ubiquitination and subsequent degradation Starch branching enzymes have been observed to interact with Pho1 in a phosphorylation-dependent manner in the amyloplast, indicating that Pho1 is involved in starch synthesis d with lower affinity toward Glc-1-P (d with proteolytic fragments (F50s) might mainly play a role in the phosphorolysis direction.A handful of proteins regulated by the proteasome are cleaved internally and only partially processed. The NF-kappaB1 precursor p105 was cleaved into a functional p50 product by the 20S proteasome  Glc-1-P . Importad) purified from heat-treated sweet potato roots displayed lower affinity toward Glc-1-P  pathway inside the chloroplast at night It is well established that the 26S proteasome is essential for the degradation of misfolded proteins and the elevated levels of the free 20S proteasome lead to increased tolerance to oxidative stress  Glc-1-P , suggestOxidatively modified proteins can form large aggregates because of increased surface hydrophobicity. Most oxidatively damaged proteins appear to undergo selective proteolysis, primarily by the proteasome. Contrary to earlier popular belief that most proteasomal degradation is conducted by the 26S proteasome with substrate ubiquitination, it has been shown that oxidized proteins are degraded without ubiquitin conjugation (or ATP hydrolysis) possibly by the 20S proteasome, and this degradation is still inhibited by proteasome inhibitors It was proposed that the hydrophobic patches of oxidized proteins bind to the α-subunits of the 20S proteasome, thereby opening the substrate channel Ipomoea batatas (L.) Lam. cv Tainong 57] were collected from a local farm near Taipei, Taiwan. Chemicals were purchased from Sigma-Aldrich Co. unless otherwise noted.Mature sweet potato roots [Protein content was determined by the dye-binding method SDS-PAGE and native PAGE were performed according to the method described by Laemmli Samples were first separated on a 6% native PAGE. After electrophoresis, the gel was incubated with the reaction buffer containing 20 mM MES-KOH (pH 5.9), 1.2% soluble starch  and 32 mM Glc-1-P g for 30 min. The supernatant was separated on a Sephacryl S-300 column at 4°C. The fractions were separated by 6% native PAGE and analyzed by Coomassie staining, Pho1 activity staining, or western blotting with α20S antibody. The chromatography column was calibrated by using gel filtration standard (Bio-Rad), containing thyroglobulin (bovine) 670 kDa, γ-globulin (bovine) 158 kDa, ovalbumin (chicken) 44 kDa, myoglobin (horse) 17 kDa, and vitamin B12 1.35 kDa.Sweet potato roots (150 g) were homogenized with buffer A  containing 1% polyvinylpolypyrrolidone in a Waring blender followed by centrifugation at 48,000 xSweet potato roots (500 g) were homogenized with buffer A  containing 1% polyvinylpolypyrrolidone in a Waring blender. The crude extract was separated on a Sephacryl S-300 column as described previously. The fractions containing Pho1 activities were determined by in-gel Pho1 activity assay. The high molecular weight fractions with Pho1 activity (HX) were collected and loaded on a DEAE-Sephacel column following the elution step with a linear gradient of NaCl (0.15∼0.5 M) in buffer A. The fractions containing HX were collected and concentrated by ultrafiltration . All purification procedures were carried out at 4°C.www.matrixscience.com).Selected protein targets were excised from the Coomassie-stained gel according to the standard MS sample preparation protocol Pho1 was purified according to a method described previously The conventional rabbit antiserum against the 20S proteasome (α20S) was generated by immunizing rabbits with the purified 20S proteasome from sweet potato roots. The mouse conventional antiserum against Pho1 was produced from mice immunized with purified Pho1 from sweet potato roots. Mouse monospecific antibody anti-L1 against the peptide sequence TKPKETSIVDPSEEVEVSG on the L78 insertion, and mouse monoclonal antibodies H7c and J3b, were prepared as described by Chen et al. Antibody crosslinking procedures were performed according to the method described by Sisson and Castor The first-dimension 6% native PAGE (0.75 mm thick) was run on a conventional slab gel system. The lanes on the gel slab were cut into individual strips, and then transferred to PVDF membranes for western blotting with specific antibodies. Alternatively, the strips were incubated with equilibration buffer  at 37°C for 30 min. Subsequently, the gel strips were placed onto the top of the stacking gel (4%) of the second-dimension 12.5% SDS-PAGE (1 mm thick). The electrophoresis was run at 150 V for 2 h. Proteins on the slab were transferred to PVDF membranes and immunostained with specific antibodies.3VO4, 1% polyvinylpolypyrrolidone and 1 mM phenylmethylsulfonyl fluoride).Sweet potato root discs (5 cm in diameter and approximately 0.5 mm thick) were cut from mature sweet potato roots in the laminar flow hood. The discs were first incubated with or without 100 µM MG132 (Sigma-Aldrich Co.) in 10 mM sterile MES-KOH (pH 6.5) buffer containing 60 mM sucrose at room temperature for 2 h. After the pretreatment, the discs were sealed in sterile plastic bags and incubated at 45°C in an oven for the indicated time intervals. At each time point, the discs were transferred quickly from the oven to the liquid nitrogen and then ground into powder in a mortar. The ground powder (0.4 g) was extracted with 200 µl of extraction buffer (100 mM Tris-HCl (pH 7.4), 5 mM EDTA, 10 mM β-mercaptoethanol, 20 mM NaF, 1 mM NaChymotrypsin-like activity of the 20S proteasome was determined by measuring the hydrolysis of the fluorogenic substrate N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin  in 100 mM Tris (pH 7.6), 1 mM DTT, 0.1 mM Suc-LLVY-AMC and 0.02% SDS at 37°C for 30 min Figure S1Characterization of intact Pho1 (Pho1) and proteolytic modified Pho1 (Pho1d) by SDS-PAGE and western blot analysis. Intact Pho1 (Pho1) was purified from sweet potato root discs which did not undergo heat treatment. Proteolytic modified Pho1 (Pho1d) was purified from sweet potato root discs which were incubated at 45°C for 36 h. Purified Pho1 or Pho1d (10 µg) was separated by 12.5% SDS-PAGE and analyzed by Coomassie Brilliant Blue R-250 (Coomassie) staining or western blot with H7c, J3b or αL78 antibody, respectively. P110 is the intact form of Pho1 revealing the molecular weight at 110 kDa. F50s are a group of proteolytic modified Pho1 revealing the molecular weight around 50 kDa. P110 was not observed in the Pho1d sample. Since αL78 did not display any cross-reaction with Pho1d, it reveals that Pho1d does not contain the L78 insertion.(DOC)Click here for additional data file.Figure S2Determination of the enzyme kinetic parameters of intact Pho1 (Pho1) and proteolytic modified Pho1 (Pho1d). (A) The double reciprocal plot using Glc-1-P (9.4 mM) as the limiting substrate. (B) The double reciprocal plot using soluble starch (0.35%) as the limiting substrate. Pho1, open circle (○); Pho1d, solid triangle (▴). Values are mean ± S.D. from three independent experiments.(DOC)Click here for additional data file.Figure S3Multiple alignment of the L78 amino acid sequences from different plant species. Multiple alignment of the L78 amino acid sequences of Pho1 from Ipomoea batatas , Solanum tuberosum , Oryza sativa  and Zea mays  was done by using ClustalW program. Identical residues conserved in all sequences were marked with asterisks (*). The conserved substitutions among different sequences were denoted as colons (:).(DOC)Click here for additional data file.Figure S4Pho1 in potato tubers undergoes proteolytic degradation upon heat treatment. Potato tubers discs (2 mm thickness) were incubated at 45°C for the indicated time periods up to 48 h. (A) The crude protein extracts of the discs (10 µg) were separated by 7.5% native PAGE and analyzed by Coomassie staining, Pho1 activity staining or western blot analysis with αPho1 polyclonal antibody, respectively. (B) Alternatively, the same samples above were subjected to 12.5% SDS-PAGE and analyzed by Coomassie or western blot analysis with αPho1, respectively.(DOC)Click here for additional data file.


Antibiotic drug discovery is an indispensable process to combat aggressive ability of pathogenic microorganisms and emerging infectious diseases against health and well-being of people throughout the world. According to the updated and expanded data on natural products presented by Newman and Cragg , the utiStreptomyces griseus there has been a rapid escalation in antibiotic discovery from the genus Streptomyces (largest genus of Actinobacteria). Subsequently, members of this genus have become renowned as a prime source of natural antibiotics  are currently isolated from diverse environments and intensively studied for potential leads for antibiotic discovery programs. The rare actinomycetes are usually regarded as strains of actinomycetes whose isolation frequency by conventional methods is much lesser than that of streptomycete strains  apparently opine that these organisms nourish the current antibiotic drug discovery programs.


Background. In this study, we examined whether UV-visible and fluorescence spectroscopy techniques detect the progression of atherosclerosis in serum of rabbits fed on high-cholesterol diet (HCD) and HCD supplemented with zinc (HCD + Zn) compared with the control. Methods. The control rabbits group was fed on 100 g/day of normal diet. The HCD group was fed on Purina Certified Rabbit Chow supplemented with 1.0% cholesterol plus 1.0% olive oil (100 g/day) for the same period. The HCD + Zn group was fed on normal Purina Certified Rabbit Chow plus 1.0% cholesterol and 1.0% olive oil supplemented with 470 ppm Zn for the same feeding period. UV-visible and fluorescence spectroscopy and biochemistry in Rabbit's blood serum and blood hematology were measured in Rabbit's blood. Results. We found that the fluorescent peak of HCD shifted toward UV-visible wavelength compared with the control using fluorescent excitation of serum at 192 nm. In addition, they showed that supplementation of zinc (350 ppm) restored the fluorescent peak closely to the control. By using UV-visible spectroscopy approach, we found that the peak absorbance of HCD (about 280 nm) was higher than that of control and that zinc supplementation seemed to decrease the absorbance. Conclusions. This study demonstrates that ultraviolet-visible and fluorescence spectroscopy techniques can be applied as noninvasive techniques on a sample blood serum for diagnosing or detecting the progression of atherosclerosis. The Zn supplementation to rabbits fed on HCD delays or retards the progression of atherosclerosis. Inducing anemia in rabbits fed on HCD delays the progression of atherosclerosis. Atherosclerosis is the primary cause of coronary and cardiovascular diseases. Atherosclerosis and heart diseases are major causes of morbidity and mortality in adults in industrialized nations. Atherosclerosis, a stenotic lesion of arterial walls, is a leading cause of death affecting almost one third of humans in developed countries. It can generally be viewed as a form of chronic inflammation that is induced and perturbed by lipid accumulation , 2. HypeAtherosclerotic lesions are characterized by progressive thickening of the arterial intima layer due to cellular proliferation (foam cells), lipid and cholesterol deposition. Following extracellular lipid accumulation, there is a change in elastin layer and enlargement of upper intima with development of a “collagenous cap” or fibroatheroma plaque. In advanced lesions lipid and necrotic deposits accumulate within the intima, and calcium crystals build-up takes place, leading to the ultimate calcification of artery wall. Although many therapies are available for treatment, usually the physician's intervention occurs when the plaque is symptomatic, with high commitment of important organs that need high blood supply, such as heart and brain –7. in vivo and potentially guiding laser angiosurgery catheters [Fluorescence spectroscopy has been recently explored as a new technique for the diagnosis of disease in human tissue –11. Seveatheters –10. Simiatheters  on the bIt has been reported that feeding rabbits a high-cholesterol diet resulted in alterations in prooxidant-antioxidant status in several tissues as well as typical atherosclerotic changes in the aorta –16.Fluorescence is the emission of light by a substance following absorption of light or other electromagnetic radiation of a different wavelength. In most cases, the emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation. However, when the absorbed electromagnetic radiation is intense, it is possible for one molecule to absorb two photons; this two-photon absorption can lead to emission of radiation having a shorter wavelength than the absorbed radiation.The Fourier-Transform Raman (FT-Raman) spectroscopy is an optical tool which permits less invasive and nondestructive analysis in biological samples with high precision, allowing one to get precision information of biochemical composition from different types of human tissues such as coronary arteries, lung, and colon without tissue removal –19. The Currently there is no noninvasive clinical method available for detecting or diagnosing atherosclerotic plaque content without withdrawing tissue. The ultraviolet-visible and fluorescence spectroscopy techniques have not been well documented in rabbits blood fed on high-cholesterol diet (HCD) and HCD supplemented with high zinc (Zn) level. Thus, the aim of this study is to diagnosis or detect the progression of atherosclerosis as well as the delay or retard of the progression of atherosclerosis applying ultraviolet-visible and fluorescence spectroscopy techniques on rabbit's blood sample. For this reason, to diagnose atherosclerosis with noninvasive techniques, a group of rabbits are fed on HCD for a feeding period of 12 weeks, and rabbits are fed on HCD supplemented with high Zn level for the same feeding period of time; then ultraviolet-visible and fluorescence spectroscopy techniques will be applied as noninvasive techniques on rabbit's blood sample.n = 8), the 2nd group is high-cholesterol diet  group, and the 3rd group is the HCD group supplemented with high Zn level (n = 8). The control group (n = 5) was fed on 100 g/day of NOR diet  for a feeding period of 12 weeks. Chemical composition of the laboratory NOR rabbit diet (Purina Certified Rabbit Chow # 5321) [n = 5) was fed on NOR Purina Certified Rabbit Chow no. 5321 supplemented with 1.0% cholesterol plus 1.0% olive oil (100 g/day) for the same feeding period of time. The HCD + Zn group was fed on NOR Purina Certified Rabbit Chow plus 1.0% cholesterol and 1.0% olive oil supplemented with 350 ppm Zn  for the same feeding period of time. The animals were sacrificed by intravenous injection of Hypnorm (0.3 mL/kg) in accordance with the guidelines approved by King Saud University Local Animal Care and Use Committee.The atherosclerotic model used in this study was the New Zealand white rabbit , obtained from the Laboratory Animal Center . Fifteen rabbits were individually caged and divided into the following. The 1st group is control group ( # 5321) –6. The HBlood samples were collected from control, HCD, and HCD + Zn groups of animals, following an overnight fast, and collectively analyzed. Blood samples of 2 mL were obtained from the rabbits via vein puncture of an antecubital vein. The blood was collected into two polypropylene tubes, one for serum and one for plasma. The blood for plasma was collected in heparin. The serum was prepared by allowing the blood to clot at 37°C and centrifuge at 3000 rpm for 10 min.Rabbit blood serum absorbance of control, HCD, and HCD + Zn groups was measured using a UV-VIS double beam spectrophotometer  through optical resolution of 0.4 nm. Absorbance measurements were obtained over wavelength range of 200–800 nm at room temperature using quartz cuvettes (1 cm path length). The cuvettes were cleaned before each use by sonicating for 5 min in deionized water followed by rinsing with deionized water. The pH for different blood serum samples was kept constant during the measurements.Fluorescence measurements were performed in rabbit's blood serum samples using a FluoroMax-2 JOBAN YVON-SPEX, Instruments S.A., Inc., France. The fluorescence measurements were made over wavelength range of 190–400 nm and at 2 different excitation values (192 and 278 nm) on series of samples with a 10 mm light path cuvette.Blood samples (2 mL) were obtained from the rabbits via vein puncture of an antecubital vein. Blood was collected into two polypropylene tubes, namely, one for serum and one for plasma. The blood for plasma was collected in heparin. Serum was prepared by allowing the blood to clot at 37°C and centrifugation at 3000 rpm for ten minutes. A hematological autoanalyzer  was used to determine different hematological and dimensional parameters, such as red blood cells (RBC), white blood cells (WBCs), hemoglobin (HB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), neutrophils%, lymphocytes%, monocytes%, eosinophils%, basophils%, mean platelet volume (MPV), platelet distribution width (PDW)%, plateletcrit (PCT)%, and platelets (PLTs). Serum TC and TG levels were analyzed by the enzymatic method used in the clinical laboratory center of King Khaled Hospital. HDL concentration was determined by the previously reported method .Animals were weighted and examined daily for health and behavioral changes following HCD and HCD + Zn administration. To evaluate the morphological changes, several samples from different rabbit organs  were cut rapidly, fixed in neutral buffered formalin (10%), and dehydrated through a series of ethanol grades . The rabbit samples were then cleared in 2 changes of xylene, impregnated with 2 changes of molten paraffin wax, embedded, and blocked. Paraffin sections (4-5 um) were stained with hematoxylin and eosin according to . Traditional histopathological technique was performed. Hematoxylin and eosin, Verhoeff, Masson, and Picrosirius staining were used for tissue structure observation. The histopathological classification was made by a board certified pathologist. We expressed the results for UV-Visible and fluorescence spectroscopy, blood biochemistry, and hematology as mean ± standard error (Mean ± SE). To assess the significance of the differences between control, HCD, and HCD + Zn, we performed the statistical analysis using one-way analysis of variance (ANOVA) for repeated measurements, with significance assessed at 5% confidence level.To diagnose or detect and delay the progression of atherosclerosis, noninvasive techniques such as ultraviolet-visible and fluorescence spectroscopy techniques were applied on rabbit's blood sample fed on HCD and HCD supplemented with high Zn level for a feeding period of 12 weeks. The normal blood is characterized by Five hemoglobin (Hb) peaks: the peak at 280 nm corresponds to the aromatic amino acids, at 340 nm corresponds to globin-heme interaction, at 420 nm corresponds to the heme, the peak at 540 nm corresponds to heme-heme interactions, and at 578 nm corresponds to hemoglobin oxygen affinity . N = 5). In each group, the absorbance peak for rabbits fed on HCD significantly increased compared with the control, while it returned towards the control absorbance peak for rabbits fed on HCD + Zn. The absorbance peak for control was significantly different from those for HCD and HCD + Zn.Figures We have observed two peaks at wavelengths 195 nm and 280 nm using the UV-Visible spectroscopy , so we have provided the average fluorescent peak value of each group (N = 5) in terms of mean ± SD, and the statistical analysis between control, HCD, and HCD + Zn was statistically significant.In this study, we have observed the same results in each group  to show the degree of oxidation in rabbit serum, which will support and link the UV-Visible, fluorescence spectroscopy data, and histopathological data in each group. In addition, we will examine the degree of atherosclerosis in the aorta by measuring the intimal and media thickness of the aorta and expressing it in terms of intimal/media ratio. Finally, we will perform UV-Visible and fluorescence spectroscopy of rabbit serum at different time points  instead of only one time point (12 weeks), after feeding with HCD to monitor whether the UV-Vis/fluorescent spectroscopy data correlate with the progression of atherosclerosis. To diagnose or detect and delay the progression of atherosclerosis, noninvasive techniques such as ultraviolet-visible and fluorescence spectroscopy techniques were applied on blood sample of rabbits fed on HCD and HCD supplemented with high Zn level for a feeding period of 12 weeks.Hypercholesterolemia can increase the cholesterol content of platelets, polymorphonuclear leukocytes, and endothelial cells so that endothelial and smooth muscle cells, neutrophils and platelets may be sources of oxygen-free radicals. It becomes evident that oxidative stress occurs when there is an excessive production of free radicals in the face of defective antioxidant defenses. At excitation of 192 nm, the fluorescence peak shifted towards the visible region for HCD group, while it returned towards the control fluorescence peak for rabbits fed on HCD + Zn. The fluorescence emission from normal rabbit blood serum was significantly different from those for HCD and HCD + Zn. At excitation of 278 nm, the fluorescence peak for normal rabbit blood serum was significantly different from those for HCD and HCD + Zn.The absorbance peak for rabbits fed on HCD significantly increased compared with the control, while it returned towards the control absorbance peak for rabbits fed on HCD + Zn. The absorbance peak for control was significantly different from those for HCD and HCD + Zn. Proteins are dynamic and essential to their function. An increase in absorbance at 280 nm might indicate abnormal protein motion and reflects a deviation from the normal structure and function. The extent of deviation reflects the degree of globin unfolding and random motion of Hb molecule under different levels of oxidative stress.This study demonstrates that ultraviolet-visible and fluorescence spectroscopy techniques can be applied as noninvasive techniques on a sample blood serum for diagnosing or detecting the progression of atherosclerosis. The Zn supplementation to rabbits fed on HCD delays or retards the progression of atherosclerosis. Inducing anemia in rabbits fed on HCD delays the progression of atherosclerosis. These conclusions might be supported by further histological investigations in rabbit's different organs.