Meta-Analysis To Test the Association of HIV-1 nef Amino Acid Differences and Deletions with Disease Progression

Previous relatively small studies have associated particular amino acid replacements and deletions in the HIV-1 nef gene with differences in the rate of HIV disease progression. We tested more rigorously whether particular nef amino acid differences and deletions are associated with HIV disease progression. Amino acid replacements and deletions in patients'' consensus sequences were investigated for 153 progressor (P), 615 long-term nonprogressor (LTNP), and 2,311 unknown progressor sequences from 582 subtype B HIV-infected patients. LTNPs had more defective nefs (interrupted by frameshifts or stop codons), but on a per-patient basis there was no excess of LTNP patients with one or more defective nef sequences compared to the Ps (P = 0.47). The high frequency of amino acid replacement at residues S⁸, V¹⁰, I¹¹, A¹⁵, V⁸⁵, V¹³³, N¹⁵⁷, S¹⁶³, V¹⁶⁸, D¹⁷⁴, R¹⁷⁸, E¹⁸², and R¹⁸⁸ in LTNPs was also seen in permuted datasets, implying that these are simply rapidly evolving residues. Permutation testing revealed that residues showing the greatest excess over expectation (A¹⁵, V⁸⁵, N¹⁵⁷, S¹⁶³, V¹⁶⁸, D¹⁷⁴, R¹⁷⁸, and R¹⁸⁸) were not significant (P = 0.77). Exploratory analysis suggested a hypothetical excess of frameshifting in the regions ⁹SVIG and ¹¹⁸QGYF among LTNPs. The regions V¹⁰ and ¹⁵²KVEEA of nef were commonly deleted in LTNPs. However, permutation testing indicated that none of the regions displayed significantly excessive deletion in LTNPs. In conclusion, meta-analysis of HIV-1 nef sequences provides no clear evidence of whether defective nef sequences or particular regions of the protein play a significant role in disease progression.HIV-infected people can be categorized according to the number of years in which they progress to AIDS. Long-term nonprogressors (LTNPs) do not progress to AIDS even after more than 10 years of infection, and they maintain stable CD4 lymphocyte counts (5, 8). Nonprogression status may reflect differences in either in the host, in viral genetics, or in environmental factors. Within the virus, R⁷⁷Q, a mutation in the HIV-1 vpr gene, was associated with both LTNP infection and impaired induction of apoptosis (38). However, this mutation was not statistically significant, and no other clearly attenuating mutations or deletions were detected (20). Most attention, however, has focused on role of the viral nef protein.In rhesus monkeys infected with simian immunodeficiency virus (SIV), a model for studying AIDS pathogenesis (37), animals infected with nef-deficient SIV showed an attenuated course of infection (17, 30, 31, 51). nef was also a major determinant of pathogenicity in transgenic mice with AIDS-like symptoms induced by HIV-1 (27). Some patients with LTNP strains of HIV were found to have gross deletions in the nef gene (16, 33, 49), suggesting the importance of nef for HIV-1 progression in humans.Previous studies related to phylogenetic analysis have reported that nef sequences from patients with different rates of progression do not form distinct clusters (28, 29, 40, 43). Each patient had sequences that clustered together and could be differentiated from those of the other patients, supporting the monophyletic origin of the infections. The absence of intragroup clustering suggested that no correlation existed between the phylogenetic relationship of the nef sequences and the progression rate in the patients (10). The differences in genetic distance between LTNP and progressors (Ps) were not statistically significant, suggesting that the degree of sequence variation in nef is unlikely to reflect the stage of HIV-1 disease (4).Amino acids 25 to 36 in HIV-1 nef are important both for several well-defined in vitro functions of nef and for the pathogenicity of HIV-1 in humans, and nef''s ability to enhance virion infectivity was fully restored when the deletion was repaired by the insertion of that region (8). Nef proteins derived from LTNPs and slow progressors (SPs) were found to be defective or far less capable of enhancing viral replication and/or viral infectivity in herpesvirus saimiri-transformed human T cells and peripheral blood mononuclear cells (PBMC) (24). The sizes of the deletions in the nef/LTR (long terminal repeat) region increased progressively from 84 to 1,400 bp during the 5-year follow-up period in one case of a SP (35). Gross defects were also present in the RNA-derived sequences of an LTNP individual because of a frameshift and the premature termination of the protein (4). HIV-1 sequences from the isolates or patient PBMC had similar deletions in the nef gene and in the region of overlap of nef and the U3 region of the LTR (16). There was a 36-bp deletion close to the 5′ end of nef that impaired nef function in an LTNP (8).Many studies not only have described nef as carrying large deletions in LTNPs (16, 33) but also found a higher proportion of disrupted nef gene sequences in LTNPs. A study of six HIV patients who reached at least 11 years of age without or with mild symptoms revealed that LTNPs had higher proportions of disrupted nef sequences (10). Seven LTNPs, all belonging to the same cohort of infected hemophiliacs, had more defective nef sequences than in progressors; the number of disrupted nef sequences within each individual was significantly higher in LTNPs than in progressors (4).The nef amino acid sequence has been reported to be highly polymorphic even within a particular subtype (4, 22, 28, 29, 40, 42, 53). Single amino acid deletions have been found predominantly at three locations that are structurally less defined loop regions: positions 8 to 11, 49 to 51, and 155 to 162 (25). Five variants (T¹⁵, N⁵¹, H¹⁰², L¹⁷⁰, and E¹⁸²) have been noted among LTNPs, whereas nine variants (N-terminal PxxP motif; A¹⁵, R³⁹, T⁵¹, T¹⁵⁷, C¹⁶³, N¹⁶⁹, Q¹⁷⁰, and M¹⁸²) have been noted among progressors (32). nef has been often changed at residues localized in the folded core domain at cytotoxic-T-lymphocyte epitopes (E¹⁰⁵, K¹⁰⁶, E¹¹⁰, Y¹³², K¹⁶⁴, and R²⁰⁰); moreover, LTNP-associated variations occur in the core domain of nef. Recently, nef sequence variations have been found in the WL motif of the CD4 binding site, as well as a premature stop codon in infected LTNPs that could potentially contribute to the attenuation of the virus; however, these deletions were found to be insignificant (13).There has been a broad agreement that grossly defective nefs are associated with an attenuated course of infection (17, 30, 31, 51) but rare in HIV-1 infection (32). Grossly defective nef genes or significant changes from relevant clade reference sequences were not identified in a study of 32 LTNP children (13). One study noted that the proportion of disrupted nef sequences within each patient was significantly higher in LTNPs compared to Ps; however, the proportions of individuals with nef defects (in LTNPs, 5 of 7, and in Ps, 6 of 8) were similar (4). No major defects have been reported in a few other studies (28, 39, 40). Another study of a small number of patients does not indicate that gross deletions play any major role in delaying or halting disease progression in infected drug abusers in Italy (11), and premature stop codons were observed at equivalent, yet low, frequencies among the different clinical groups (41). In addition, disease progression has been reported in a HIV patient with a virus grossly deleted of nef (26).Thus, overall, most of these studies were based on observation or case study rather than systematic scientific evaluation (11). The objective of the present study was to determine in a substantially larger sample than investigated to date whether there is any association between disease progression and particular nef amino acid differences or deletions.     

不同病因的慢性肾病患者血清氨基酸谱存在显著差异

慢性肾病(chronic kidney disease, CKD)患者血清氨基酸谱发生了显著性变化,但目前并无不同病因的CKD患者血清氨基酸谱的比较研究。本研究主要探究不同病因CKD患者血清氨基酸谱的差异,以及差异氨基酸与肾疾病发生发展的相互关系。选取79例确诊慢性肾病的成年患者,根据其病因,分糖尿病肾病组、高血压肾病组及慢性肾小球肾炎组。另选取25例性别年龄匹配的健康成年人为正常对照组。收集及处理清晨空腹血清标本,采用反相高效液相色谱法（reverse-phase high-performance liquid chromatography, RP-HPLC）测定血清中22种游离氨基酸的水平。结果显示,与正常对照组相比,3组CKD患者血清赖氨酸、丝氨酸、甘氨酸、伽马氨基丁酸(GABA)、色氨酸、亮氨酸以及酪氨酸水平均显著下降（P<0.05）,而血氨水平显著升高（P<0.05）。糖尿病肾病组患者血清苏氨酸水平明显高于其余3组（P<0.05）,而血清天冬氨酸水平显著低于其余3组（P<0.05）。慢性肾小球肾炎患者血清异亮氨酸水平显著低于糖尿病肾病及高血压肾病患者（P<0.05）。上述结果证实,慢性肾病患者血清氨基酸谱较正常对照发生显著变化,且不同病因CKD患者部分血清氨基酸水平存在显著差异。其中,色氨酸水平的差异可能是不同病因CKD患者肾功能恶化速度不一致的原因之一。     

Metabolite Profiles of Lactic Acid Bacteria in Grass Silage

The metabolite production of lactic acid bacteria (LAB) on silage was investigated. The aim was to compare the production of antifungal metabolites in silage with the production in liquid cultures previously studied in our laboratory. The following metabolites were found to be present at elevated concentrations in silos inoculated with LAB strains: 3-hydroxydecanoic acid, 2-hydroxy-4-methylpentanoic acid, benzoic acid, catechol, hydrocinnamic acid, salicylic acid, 3-phenyllactic acid, 4-hydroxybenzoic acid, (trans, trans)-3,4-dihydroxycyclohexane-1-carboxylic acid, p-hydrocoumaric acid, vanillic acid, azelaic acid, hydroferulic acid, p-coumaric acid, hydrocaffeic acid, ferulic acid, and caffeic acid. Among these metabolites, the antifungal compounds 3-phenyllactic acid and 3-hydroxydecanoic acid were previously isolated in our laboratory from liquid cultures of the same LAB strains by bioassay-guided fractionation. It was concluded that other metabolites, e.g., p-hydrocoumaric acid, hydroferulic acid, and p-coumaric acid, were released from the grass by the added LAB strains. The antifungal activities of the identified metabolites in 100 mM lactic acid were investigated. The MICs against Pichia anomala, Penicillium roqueforti, and Aspergillus fumigatus were determined, and 3-hydroxydecanoic acid showed the lowest MIC (0.1 mg ml⁻¹ for two of the three test organisms).     

Effects of Ethylene Glycol Monomethyl Ether and Its Metabolite, 2‐Methoxyacetic Acid,on Organogenesis Stage Mouse Limbs In Vitro

Exposure to ethylene glycol monomethyl ether (EGME), a glycol ether compound found in numerous industrial products, or to its active metabolite, 2‐methoxyacetic acid (2‐MAA), increases the incidence of developmental defects. Using an in vitro limb bud culture system, we tested the hypothesis that the effects of EGME on limb development are mediated by 2‐MAA‐induced alterations in acetylation programming. Murine gestation day 12 embryonic forelimbs were exposed to 3, 10, or 30 mM EGME or 2‐MAA in culture for 6 days to examine effects on limb morphology; limbs were cultured for 1 to 24 hr to monitor effects on the acetylation of histones (H3K9 and H4K12), a nonhistone protein, p53 (p53K379), and markers for cell cycle arrest (p21) and apoptosis (cleaved caspase‐3). EGME had little effect on limb morphology and no significant effects on the acetylation of histones or p53 or on biomarkers for cell cycle arrest or apoptosis. In contrast, 2‐MAA exposure resulted in a significant concentration‐dependent increase in limb abnormalities. 2‐MAA induced the hyperacetylation of histones H3K9Ac and H4K12Ac at all concentrations tested (3, 10, and 30 mM). Exposure to 10 or 30 mM 2‐MAA significantly increased acetylation of p53 at K379, p21 expression, and caspase‐3 cleavage. Thus, 2‐MAA, the proximate metabolite of EGME, disrupts limb development in vitro, modifies acetylation programming, and induces biomarkers of cell cycle arrest and apoptosis     

Moisture Requirements for Growth and Metabolite Production by Lactic Acid Bacteria

The effect of water activity (a_w) reduction on growth and acid and diacetyl production by three lactic streptococci was studied. In addition, the influence of low moisture conditions on several bacteria of significance in the fermentation of sauerkraut was examined. The minimal a_w supporting growth of dairy lactics was 0.93 in a medium adjusted with glycerol. Media adjusted with sucrose generally were more inhibitory than those in which glycerol was the humectant. Titratable acidity, although not related to the type of humectant, did depend on the a_w of the medium and was directly related to the extent of growth. Diacetyl concentration increased in cultures of reduced a_w when the media were adjusted with both humectants; however, the effect was greatest with glycerol. A lactic strain associated with sauerkraut fermentation appeared to grow at a lower minimal a_w in a glycerol-adjusted medium than in a system adjusted with NaCl; however, none of the sauerkraut organisms grew at a_w levels of <0.95 when NaCl was the solute. Acid production appeared to be related to the presence and extent of growth at all of the a_w levels studied.     

Comparative Metabolite Profiling of Two Rice Genotypes with Contrasting Salt Stress Tolerance at the Seedling Stage

Background

Rice is sensitive to salt stress, especially at the seedling stage, with rice varieties differing remarkably in salt tolerance (ST). To understand the physiological mechanisms of ST, we investigated salt stress responses at the metabolite level.

Methods

Gas chromatography-mass spectrometry was used to profile metabolite changes in the salt-tolerant line FL478 and the sensitive variety IR64 under a salt-stress time series. Additionally, several physiological traits related to ST were investigated.

Results

We characterized 92 primary metabolites in the leaves and roots of the two genotypes under stress and control conditions. The metabolites were temporally, tissue-specifically and genotype-dependently regulated under salt stress. Sugars and amino acids (AAs) increased significantly in the leaves and roots of both genotypes, while organic acids (OAs) increased in roots and decreased in leaves. Compared with IR64, FL478 experienced greater increases in sugars and AAs and more pronounced decreases in OAs in both tissues; additionally, the maximum change in sugars and AAs occurred later, while OAs changed earlier. Moreover, less Na⁺ and higher relative water content were observed in FL478. Eleven metabolites, including AAs and sugars, were specifically increased in FL478 over the course of the treatment.

Conclusions

Metabolic responses of rice to salt stress are dynamic and involve many metabolites. The greater ST of FL478 is due to different adaptive reactions at different stress times. At early salt-stress stages, FL478 adapts to stress by decreasing OA levels or by quickly depressing growth; during later stages, more metabolites are accumulated, thereby serving as compatible solutes against osmotic challenge induced by salt stress.     

Isolation of o-Acetylbenzene-amidinocarboxylic Acid,a New Metabolite of Gibberella saubineti

The nickel requirement and the role of nickel were investigated in a recently identified oxygen-resistant hydrogen bacterium, Xanthobacter autotrophicus strain Y38. When 0.3 μm NiSO₄ was added to the basal medium which had not been supplemented with nickel, the cell concentration of autotrophically grown strain Y38 increased by about 4-fold and the resumption of cell growth occurred in the stationary phase. These results showed the requirement of nickel for the autotrophic growth of strain Y38. Since a trace of nickel was detected in the basal medium, the role of nickel was investigated using 0.2 mm or 0.4 mm EDTA-containing media. Other trace elements, Ca, Co, Cu, Mn, Mo and Zn, could not replace nickel. Nickel was not required for the heterotrophic growth of strain Y38. Nickel seems to be related a little to urease in strain Y38. Moderate hydrogenase induction was observed in hydrogenase deficient cells of strain Y38 under 95%H₂ + 5%O₂ when 300 μm NiSO₄ was added to 0.4 mm EDTA-containing buffer but it was completely inhibited by chloramphenicol, indicating that nickel was related to the hydrogenase synthesis. A nickel dependent increase in growth rate was demonstrated equally under 40%O₂ and 10%O₂, suggesting that nickel was not directly related to the oxygen-resistance of strain Y38.     

脑胶质瘤中心体的异常扩增及其与疾病分期的相关性

目的:探讨脑胶质瘤中心体扩增情况及其与疾病分期的相关性。方法:对40例胶质瘤标本和10例正常脑组织标本进行HE染色的检测;免疫组织化学检测Ki67和γ-微管蛋白的表达;使用免疫荧光染色技术检测中心体扩增情况。结果:不同标本中,HE染色呈现不同的细胞形态特征;Ki67在正常脑组织中没有表达,但在Ⅰ/Ⅱ、Ⅲ和Ⅳ级胶质瘤中的阳性表达率分别为65%、80%和100%,各组间差异具有统计学意义(P<0.01),说明Ki67的表达与胶质瘤级别相关。γ-微管蛋白在正常脑组织和Ⅰ/Ⅱ、Ⅲ、Ⅳ级胶质瘤中的表达率分别为30%、50%、80%和100%,各组间差异具有统计学意义(P<0.01),说明胶质瘤级别升高,中心体扩增率增加;免疫荧光检测显示,中心体扩增率和脑胶质瘤的级别呈正相关。结论:中心体扩增是脑胶质瘤的恶性程度的特征之一,并且与胶质瘤发病阶段有关,提示中心体扩增和脑胶质瘤的发展具有密切的相关性。     

Metabolite activation of crassulacean Acid metabolism and c(4) phosphoenolpyruvate carboxylase

The effects of glycine, alanine, serine, and various phosphorylated metabolites on the activity of phosphoenolpyruvate (PEP) carboxylase from Zea mays and Crassula argentea were studied. The maize enzyme was found to be activated by amino acids at a site that is separate from the glucose 6-phosphate binding site. The combination of glycine and glucose 6-phosphate synergistically reduced the apparent K_m of the enzyme for PEP and increased the apparent V_max. Of the amino acids tested, glycine showed the lowest apparent K_a and caused the greatest activation. d-Isomers of alanine and serine were more effective activators than the l-isomers. Unlike the maize enzyme, the Crassula enzyme was not activated by amino acids. Activation of either the Crassula or maize enzyme by glucose 6-phosphate occurred without dephosphorylation of the activator molecule. Furthermore, the Crassula enzyme was activated by two compounds containing phosphonate groups whose carbon-phosphorus bonds were not cleaved by the enzyme. A study of analogs of glucose 6-phosphate with Crassula PEP carboxylase revealed that the identity of the ring heteroatom was a significant structural feature affecting activation. Activation was not highly sensitive to the orientation of the hydroxyl group at the second or fourth carbon positions or to the presence of a hydroxyl group at the second position. However, the position of the phosphate group was found to be a significant factor.     

Long-Chain Fatty Acid Combustion Rate Is Associated with Unique Metabolite Profiles in Skeletal Muscle Mitochondria

Background/Aim

Incomplete or limited long-chain fatty acid (LCFA) combustion in skeletal muscle has been associated with insulin resistance. Signals that are responsive to shifts in LCFA β-oxidation rate or degree of intramitochondrial catabolism are hypothesized to regulate second messenger systems downstream of the insulin receptor. Recent evidence supports a causal link between mitochondrial LCFA combustion in skeletal muscle and insulin resistance. We have used unbiased metabolite profiling of mouse muscle mitochondria with the aim of identifying candidate metabolites within or effluxed from mitochondria and that are shifted with LCFA combustion rate.

Methodology/Principal Findings

Large-scale unbiased metabolomics analysis was performed using GC/TOF-MS on buffer and mitochondrial matrix fractions obtained prior to and after 20 min of palmitate catabolism (n = 7 mice/condition). Three palmitate concentrations (2, 9 and 19 µM; corresponding to low, intermediate and high oxidation rates) and 9 µM palmitate plus tricarboxylic acid (TCA) cycle and electron transport chain inhibitors were each tested and compared to zero palmitate control incubations. Paired comparisons of the 0 and 20 min samples were made by Student''s t-test. False discovery rate were estimated and Type I error rates assigned. Major metabolite groups were organic acids, amines and amino acids, free fatty acids and sugar phosphates. Palmitate oxidation was associated with unique profiles of metabolites, a subset of which correlated to palmitate oxidation rate. In particular, palmitate oxidation rate was associated with distinct changes in the levels of TCA cycle intermediates within and effluxed from mitochondria.

Conclusions/Significance

This proof-of-principle study establishes that large-scale metabolomics methods can be applied to organelle-level models to discover metabolite patterns reflective of LCFA combustion, which may lead to identification of molecules linking muscle fat metabolism and insulin signaling. Our results suggest that future studies should focus on the fate of effluxed TCA cycle intermediates and on mechanisms ensuring their replenishment during LCFA metabolism in skeletal muscle.     

L-Phosphoserine, a Metabolite Elevated in Alzheimer''s Disease, Interacts with Specific L-Glutamate Receptor Subtypes

L-Phosphoserine is one of the phosphomonoesters elevated in Alzheimer's disease brain and has close structural similarity to L-glutamate. This study attempts to define precisely the actions of L-phosphoserine at L-glutamate receptor subtypes. L-Phosphoserine is shown to bind to N-methyl-D-aspartate and kainic acid receptor subtypes, but not to the quisqualic acid subtype. Studies of [3H]MK-801 binding in the presence and absence of L-glutamate and glycine show L-phosphoserine to be a competitive N-methyl-D-aspartate antagonist. The IC50 of L-phosphoserine in these studies varies from 373 to 721 microM. This may indicate a physiologically relevant action of L-phosphoserine in Alzheimer's disease brain because whole brain concentrations may reach over 1 mM.     

Marked Amine and Amine Metabolite Changes in Norrie Disease Patients with an X-Chromosomal Deletion Affecting Monoamine Oxidase

Urinary and plasma amines and amine metabolites were quantified in two individuals with Norrie disease resulting from a deletion in chromosomal region Xp11.3, recently reported to be associated with absence of the gene encoding monoamine oxidase (MAO)-A and nondetectable MAO-A activity in fibroblasts and MAO-B activity in platelets. Marked (four-to 100-fold) elevations in levels of urinary phenylethylamine, o-tyramine, and m-tyramine (which are preferential substrates for MAO-B) and marked reductions (90%) in levels of 3-methoxy-4-hydroxyphenylglycol (a deaminated metabolite of norepinephrine, a preferential substrate for MAO-A) in urine and plasma confirmed the presence of a systemic, functionally significant reduction in the activities of both MAO isozymes. The magnitude of these changes, which are equivalent to those found in subjects taking MAO-inhibiting antidepressants, suggests that early initiation of dietary and drug restrictions may be clinically important in these and other patients with X-chromosomal mutations involving MAO. These findings further support the proposition that the MAOA and MAOB genes are located in close proximity on the X chromosome. Negligible changes in the metabolites of dopamine and serotonin raise the possibility that other metabolic pathways are of importance for their production, that dietary or intestinal bacterial sources contribute substantially to the presence of these amine metabolites in urine, or both.     

游离脂肪酸与心血管疾病

游离脂肪酸是机体的一种重要的能量物质,其代谢异常可以敏感的反应脂类代谢异常,是糖代谢紊乱、胰岛素抵抗发生发展的重要促进因素,也与高血压的发生发展明显相关.有研究发现在冠心病患者血清游离脂肪酸含量明显升高,游离脂肪酸参与冠状动脉发生过程中的氧化应激反应,参与破坏血管内皮细胞功能,促进冠心病的发生发展,并与冠心病心律失常等各种并发症的发生相关.作为心肌细胞代谢的能量底物,游离脂肪酸代谢异常在心衰患者的心肌细胞能量代谢障碍中起重要作用,心衰患者心肌细胞的能量代谢与正常成人的心肌细胞代谢出现明显差别,游离脂肪酸利用减少,而葡萄糖利用增加,现已经有应用阿昔莫司、乙克莫舍、哌克西林、曲美他嗪等药物通过调整游离脂肪酸代谢,从而改善心衰细胞心肌能量代谢的尝试.游离脂肪酸代谢异常的调控有望成为心血管疾病及其危险因素治疗及预防的新靶点.     

Phoma Leaf Spot Disease of Tinospora Cordifolia and its Effect on Secondary Metabolite Production

Tinospora cordifolia is one of the important medicinal climbers growing extensively in Bhadra Wildlife Sanctuary, Karnataka, India. The plant foliages were found infected with Phoma putaminum in different parts of the sanctuary. A three‐year (August 2006–July 2009) study of the disease due to the pathogen indicated that the disease incidence (DI) ranged from 0 to 100% (maximum in Kakanahasudi), while disease severity (DS) ranged from 1.60 to 45.00% (maximum in Madhuguni). The environmental parameters like rainfall and relative humidity (RH) correlated significantly with DI and DS, while temperature correlated negatively. The regression analysis indicated that DI and DS were affected due to increase in RH and decrease in temperature and rainfall. The spatial heterogeneity of the foliar disease determined by the binary form of modified Taylor's power law indicated that the disease incidence showed the regular pattern of dispersion (P < 0.001) in seven forest regions and heterogenous pattern (P < 0.001) in one forest region. The result also indicated that the alkaloid content decreased drastically following infection with P. putaminum, while phenol, flavonoid and steroid contents increased with increase in the severity of infection.     

Cellular Senescence in Livers from Children with End Stage Liver Disease

Background

Senescent cells occur in adults with cirrhotic livers independent of the etiology. Aim: Investigate the presence rate of cellular senescence and expression of cell cycle check points in livers from children with end stage disease.

Methodology/Principal Findings

Livers of five children aged three years or less undergoing liver transplantation due to tyrosinemia (n = 1), biliary atresia (n = 2), or fulminant hepatitis (n = 2) were analyzed for senescence associated β-galactosidase (SA-βgal) activity and p16INK4a, p21cip1 and p53. All livers displayed positive cellular staining for SA-βgal in the canals of Hering and interlobular biliary ducts. In the presence of cirrhosis (3/5 cases) SA-βgal was found at the cholangioles and hepatocytes surrounding the regenerative nodules. Children with fulminant hepatic failure without cirrhosis had significant ductular transformation with intense SA-βgal activity. No SA-βgal activity was evident in the fibrous septa. Staining for p53 had a similar distribution to that observed for SA-βgal. Staining for p16^INK4a and p21^cip1 was positive in the explanted liver of the patient with tyrosinemia, in the hepatocytes, the canals of Hering, cholangioles and interlobular bile ducts. In the livers with fulminant hepatitis, p21^cip1 staining occurred in the areas of ductular transformation and in the interlobular bile ducts.

Conclusions/Significance

Cellular senescence in livers of children with end stage disease is associated with damage rather than corresponding to an age dependent phenomenon. Further studies are needed to support the hypothesis that these senescence markers correlate with disease progression.     

Labeling of nucleosides with fluorescamine and detection by spectrofluorometer for End Stage Renal Disease

Nucleosides are characterized as biomarkers in AIDS, Alzheimer, tumor, breast cancer and various malignant diseases. In the present work a direct method for the detection of nucleosides (adenosine, cytidine, uridine and guanosine) from urine samples has been developed. Nucleosides represent the extent of damage in genetic material, analysis of nucleosides by ultrasonic assisted microextraction effectively eliminates the interfering constituent of urine. This has made it a highly selective and sensitive method to analyze the nucleosides with a lower limit of detection 0.220 μmol/L and Limit of quantitation 0.660 μmol/L. The method has been validated with good linearity and correlation of coefficients of the calibration curves was higher than 0.997. The coefficients were in the range of 0.11–16.92% (inter-day) and 0.38–16.43% (intra-day), respectively.     

Metabolomic Analyses of Leishmania Reveal Multiple Species Differences and Large Differences in Amino Acid Metabolism

Comparative genomic analyses of Leishmania species have revealed relatively minor heterogeneity amongst recognised housekeeping genes and yet the species cause distinct infections and pathogenesis in their mammalian hosts. To gain greater information on the biochemical variation between species, and insights into possible metabolic mechanisms underpinning visceral and cutaneous leishmaniasis, we have undertaken in this study a comparative analysis of the metabolomes of promastigotes of L. donovani, L. major and L. mexicana. The analysis revealed 64 metabolites with confirmed identity differing 3-fold or more between the cell extracts of species, with 161 putatively identified metabolites differing similarly. Analysis of the media from cultures revealed an at least 3-fold difference in use or excretion of 43 metabolites of confirmed identity and 87 putatively identified metabolites that differed to a similar extent. Strikingly large differences were detected in their extent of amino acid use and metabolism, especially for tryptophan, aspartate, arginine and proline. Major pathways of tryptophan and arginine catabolism were shown to be to indole-3-lactate and arginic acid, respectively, which were excreted. The data presented provide clear evidence on the value of global metabolomic analyses in detecting species-specific metabolic features, thus application of this technology should be a major contributor to gaining greater understanding of how pathogens are adapted to infecting their hosts.