The role of cytoplasmic proteins binding to Band 3 protein has been also considered by assaying H2O2 effects on hemoglobin-free resealed ghosts of erythrocytes. Oxidative conditions were induced by 30 min exposure of human erythrocytes to different H2O2 concentrations (10 to 300 μM), with or without GSH  or curcumin (10 μM), compounds with proved antioxidant properties. Since SO4= influx through Band 3 protein is slower and better controllable than Cl- or HCO3- exchange, the rate constant for SO4= uptake was measured to prove anion transport efficiency, while MDA  levels and –SH groups were estimated to quantify the effect of oxidative stress. H2O2 induced a significant decrease in rate constant for SO4= uptake at both 100 and 300 μM H2O2. This reduction, observed in erythrocytes but not in resealed ghosts and associated to increase in neither MDA levels nor in –SH groups, was impaired by both curcumin and GSH, whereas only curcumin effectively restored H2O2-induced changes in erythrocytes shape. Our results show that: i) 30 min exposure to 300 μM H2O2 reduced SO4= uptake in human erythrocytes; ii) oxidative damage was revealed by the reduction in rate constant for SO4= uptake, but not by MDA or –SH groups levels; iii) the damage was produced via cytoplasmic components which cross link with Band 3 protein; iv) the natural antioxidant curcumin may be useful in protecting erythrocytes from oxidative injury; v) SO4= uptake through Band 3 protein may be reasonably suggested as a tool to monitor erythrocytes function under oxidative conditions possibly deriving from alcohol consumption, use of drugs, radiographic contrast media administration, hyperglicemia or neurodegenerative diseases.The aim of the present investigation was to verify the effect of H After 10 min stirring at 0°C, hemoglobin and intracellular content were eliminated by repeated centrifugations . After the last centrifugation, the supernatant was removed and replaced with 35 ml isotonic resealing medium , pre-heated at 37°C. Membranes were incubated at 37°C for 45 min, to allow resealing. Then, pink resealed ghosts, containing about 10% of the original hemoglobin, were used for SO4= uptake measurement, following the protocol above described for intact erythrocytes.Pink resealed ghosts of human erythrocytes were prepared as reported elsewhere , with sl4= transport assessment in control conditions the following experimental protocols were used:After rate constant for SO2O2 at different concentrations (10-100-300 μM respectively). After 30 min incubation at 25°C, samples were centrifuged to remove the supernatant and erythrocytes re-suspended to 3% hematocrit in SO4= medium. SO4= uptake was measured following what described for control conditions. Erythrocytes were observed under a light microscope at 5 min and 90 min of incubation in SO4= medium and damaged cells counted per microscopic field at the end of incubation.i) intact erythrocytes, after washing, were diluted to 3% hematocrit in isotonic medium plus H2O2 (data not shown), not allowing to measure SO4= uptake. On the other hand, lower concentrations were not used as ineffective. provoked, they. After 30 min incubation at 25°C, 300 μM H2O2 was added to either GSH- or curcumin-containing tube. Such treated samples were incubated for further 30 min at 25°C, before re-suspension in SO4= medium. SO4= uptake was then measured as described for control conditions and erythrocytes checked under a light microscope at 5 min and 90 min of incubation in SO4= medium. Damaged erythrocytes were quantified by cell count per microscopic field at the end of incubation.ii) intact erythrocytes, after washing, were diluted to 3% hematocrit in isotonic medium containing either 2 mM GSH or, alternatively, 10 μM curcumin , as anti2O2. After 30 min incubation at 25°C, samples were centrifuged to remove supernatant and resealed ghosts re-suspended to the same hematocrit in SO4= medium. SO4= uptake was then measured as described for control conditions.iii) Resealed ghosts of erythrocytes, once obtained, were re-suspended to 3% hematocrit in 35 ml isotonic medium plus 300 μM H2O2, TBARS (thiobarbituric acid reactive species) levels, resulting from reaction between TBA (thiobarbituric acid) and MDA , the end product of the lipid peroxidation process, were measured. One molecule of MDA and two molecules of TBA make up a chromogen-condensing product, formed in acidic environment, spectrophotometrically read at 532 nm [To evaluate the oxidation status of membrane, in both intact erythrocytes and resealed ghosts, treated or not with Ht 532 nm . In deta2O2 or 300 μM H2O2 plus antioxidants) after Band 3 protein isolation [-SH groups estimation was performed on both intact erythrocytes and resealed ghosts . After 10 min stirring at 0°C, hemoglobin and intracellular content were eliminated by repeated centrifugations . The process, for intact erythrocytes, was repeated with the same hypotonic buffer to discard hemoglobin. One volume of membranes  was then incubated with nine volumes of 0.1 M NaOH for 30 min at 0°C in presence of 200 μM DTT (dithiothreitol) and 20 μg/ml PMSF (Phenylmethylsulfonil fluoride). After incubation, samples were centrifuged . The pellet, containing Band 3 protein, was washed thrice with 5 mM sodium phosphate (pH 8.0) and used for -SH groups determination. For this purpose, pellet (200 μl) was solubilized by incubating 300 μl of 20% v/v SDS (Sodium dodecyl sulphate) reagent in 3 ml of 100 mM sodium phosphate (pH 8.0), for 30 min at 37°C. Samples were further incubated with 100 μl of 10 mM DTNB  in 100 mM sodium phosphate (pH 8.0), for 20 min at 37°C. DTNB reacts specifically with thiol groups resulting in a highly coloured yellow anion. NEM  was used as a positive control to obtain complete oxidation of –SH groups [Intact erythrocytes or, alternatively, resealed ghosts, after washing with isotonic medium (10% hematocrit), were lysed by cold hypotonic buffer -1E,6E-heptadiene-3,5-dione, MW = 368.4, “pro analysis” purity grade, Sigma, code C7727  was kindly provided by Prof. S. Cuzzocrea  and dissolved in 0.5% v/v DMSO, as stock solution. Solvents (ethanol and DMSO) were preventively tested upon erythrocytes to exclude any damage.All chemicals were purchased from Sigma . Ht test or one-way analysis of variance (ANOVA), followed by Bonferroni's post hoc test and were assumed significant at p<0.05 ; N represents the number of independent experiments. Statistical analysis, reported in both text and figure legends, refers to rate constant values for SO4= uptake, unless differently stated.All data are expressed as arithmetic means ± S.E.M. (standard error of the mean) for statistical analysis. GraphPad Prism software  was used. Statistically significant differences between means were tested by paired Student’s 4= uptake in both treated and untreated erythrocytes. Treatment with 10 μM DIDS, applied at the beginning of incubation in SO4= medium, completely blocked SO4= uptake and, hence, considered as a positive control. SO4= transport in control erythrocytes (untreated) increased steeply at the initial stage and reached equilibrium within 30 min . The rate constants for SO4= uptake, accounting for this inhibition, are reported in 4= incorporated by the cells in both control and 10 μM H2O2-treated cells was significantly higher (p<0.001) than that one measured in 100 μM H2O2, 300 μM H2O2and DIDS treatment, respectively  did not elicit any morphological change (data not shown). In control conditions  than what observed in 300 μM H2O2-treated erythrocytes , while significantly lower than that one observed in untreated erythrocytes .With regard to curcumin, treatment of erythrocytes with this natural antioxidant (10 μM), followed by exposure to 300 μM H2O2 , impaire2O2 treatment were prevented by 10 μM curcumin, at both 5 min and 90 min incubation in SO4= medium. Percentage of damaged cells after 10 μM curcumin plus 300 μM H2O2 treatment .As shown in ed cells , n.s.. T4= uptake in untreated resealed ghosts of erythrocytes. As a positive control, 10 μM DIDS was applied at the beginning of the incubation in SO4= medium. The rate constant for SO4= uptake in untreated resealed ghosts was significantly higher  than in DIDS-treated cells. The rate constants for SO4= uptake in these experimental conditions are reported in hrocytes , n.s., w2O2-treated resealed ghosts, MDA levels, as well as –SH groups quantity, were not significantly different with respect to those measured in untreated ghosts (data not shown). Also in this case, no further protocols with H2O2 treatment combined with antioxidants was used.In 300 μ M H2O2) is produced by O2.- dismutation, inducing oxidative modifications in oxyhemoglobin [2.-), the main source of ROS in erythrocytes. It has been shown that the exposure of oxyhemoglobin to H2O2 leads to increased concentration of methemoglobin, lipid peroxidation and spectrin-hemoglobin complexes [2O2 on Band 3 protein, the most abundant protein in human erythrocytes [Hydrogen peroxide  and its cross linking with cytoplasmic proteins [Resealed ghosts preparation is a validated method for drug delivery , for proproteins . The useproteins .2O2 did not inhibit the rate constant for SO4= uptake with respect to untreated ghosts, thus suggesting that the first target of H2O2 seems not to be the membrane, but the intracellular content, most likely Hb, which cross links with integral membrane protein, according to findings from other authors [2O2 treatment has been also demonstrated  and curcumin (10 μM), have been used as antioxidant compounds. With regard to GSH experiments, the H2O2-induced reduction in rate constant for SO4= uptake was recovered, though not completely restored, by pre-treatment of erythrocytes with GSH, whereas cell shape alterations, due to exposure to H2O2, were not impaired. Hence, we may suggest that GSH, in these experimental conditions, is not completely effective in protecting erythrocytes from oxidative damage, not preserving cell shape, which is indeed critical for erythrocytes function.In an attempt to protect Band 3 protein from H2O2-induced cell shape alterations, detected in our experiments. Moving from this consideration, curcumin, a natural antioxidant permeating cell membrane and already known for its beneficial properties [4= transport under H2O2-induced oxidative conditions in human erythrocytes.It has been already shown that GSH is not able to cross the plasma membrane, so that the intracellular GSH is the only source for erythrocytes ,36. Thisoperties ,37,38, h2O2-induced cell shape alterations and reduction in rate constant for SO4= uptake. Therefore, we may hypothesize that, in the present experimental conditions, this natural antioxidant seems to be more effective in protecting both anatomical and functional properties of erythrocytes, when exposed to oxidative stressors.Pre-treatment of erythrocytes with curcumin, significantly prevented both H4= transport in human erythrocytes exposed to stressors in the external medium, like acid pH, has been recently demonstrated [This result could be explained with the antioxidant properties of flavonoids. Some flavonoids may incorporate into the hydrophobic membrane bilayer, thus reducing its fluidity and stability ,40, whicnstrated , support4= uptake of Band 3 protein is protected by curcumin, when erythrocytes are exposed to H2O2 induced-oxidative stress. This is in line with what recently shown by Yang and co-authors [Here we show similar effects, by proving that not only cell shape but also the rate constant for SO-authors , reporti2O2.The beneficial action of curcumin on Band 3 protein function may also open the way to further considerations, taking into account the variety of oxidative stress sources affecting human tissues, including the above mentioned hyperglycemia and even On this basis and supported by the present results, we may underline the adjuvant role of natural antioxidants assumed with food, such as curcumin, in protecting Band 3 function from external stressors, such as increase in ROS levels, which correlate with high glucose blood levels, aging, physical exercise, use of drugs and their metabolites, neurodegenerative diseases and chronic alcohol consumption ,44,45,464= uptake measurement, is affected by low concentrated H2O2, adding more information about the effect of oxidative stress at structural level, as already assessed by other authors [2O2 induces oxidative damage not detectable by MDA assay or –SH group estimation and is sufficient to alter Band 3 protein efficiency, estimated by SO4= uptake measurement; natural antioxidants like curcumin are effective in protecting erythrocytes from this damage; Band 3 protein function assessment through SO4= uptake measurement can be confirmed as a tool to monitor oxidative stress effect on erythrocytes.Our results further confirm that, in human erythrocytes, the link between Band 3 protein and intracellular content is crucial for maintaining normal shape and, in turn, oxygen exchange efficiency; demonstrate that Band 3 protein function, monitored by SO4= transport measurement, related to hemoglobin modification [These findings open the way to further investigations, specifically addressed to evaluate, at level of erythrocytes function, the consequences of oxidative conditions possibly provoked by strenuous physical exercise, alcohol consumption, drugs administration or neurodegenerative diseases, which have nowadays become very common. Aged erythrocytes could be also considered for future study about the impact of aging on their function, monitored through SOfication ,48. The fication , would bS1 Text(PDF)Click here for additional data file.


Plasmodium vivax 48/45 protein is expressed on the surface of gametocytes/gametes and plays a key role in gamete fusion during fertilization. This protein was recently expressed in Escherichia coli host as a recombinant product that was highly immunogenic in mice and monkeys and induced antibodies with high transmission-blocking activity, suggesting its potential as a P. vivax transmission-blocking vaccine candidate. To determine sequence polymorphism of natural parasite isolates and its potential influence on the protein structure, all pvs48/45 sequences reported in databases from around the world as well as those from low-transmission settings of Latin America were compared.Plasmodium vivax parasite isolates from malaria-endemic regions of Colombia, Brazil and Honduras (n = 60) were used to sequence the Pvs48/45 gene, and compared to those previously reported to GenBank and PlasmoDB (n = 222). Pvs48/45 gene haplotypes were analysed to determine the functional significance of genetic variation in protein structure and vaccine potential.Nine non-synonymous substitutions  and three synonymous substitutions  that define seven different haplotypes were found among the 282 isolates from nine countries when compared with the Sal I reference sequence. Nucleotide diversity (π) was 0.00173 for worldwide samples (range 0.00033–0.00216), resulting in relatively high diversity in Myanmar and Colombia, and low diversity in Mexico, Peru and South Korea. The two most frequent substitutions  were predicted to be located in antigenic regions without affecting putative B cell epitopes or the tertiary protein structure.pvs48/45 with noted geographical clustering among Asian and American isolates. The low genetic diversity of the protein does not influence the predicted antigenicity or protein structure and, therefore, supports its further development as transmission-blocking vaccine candidate.There is limited sequence polymorphism in  Plasmodium which is transmitted by bites of infected Anopheles mosquitoes. Plasmodium falciparum and Plasmodium vivax are the two most common malaria parasites in humans, however differing in their clinical presentation and geographic distribution. Plasmodium falciparum causes the most severe symptoms and higher mortality, mainly among children under 5 years of age in Africa. Plasmodium vivax generally causes milder disease, is significantly less life-threatening [Malaria is an infectious parasitic disease caused by the genus eatening  and is weatening . Despiteeatening , 4.Proteins expressed by parasite sexual stages, namely gametocytes/gametes, could induce effective immune responses in the human host that would prevent gamete fertilization and zygote formation when ingested by the mosquito during a blood meal .PlasmodiumPs48/45 proteins are expressed by male and female gametocytes/gametes during the parasite maturation process, and are therefore classified as pre-fertilization antigens [Plasmodium species characterized by the presence of partially conserved domains containing six cysteine (Cys) amino acid residues that form one to three disulfide bridges, resulting in a specific tertiary structure [P. falciparum, this protein (Pfs48/45) is expressed on the surface membrane of gametocytes [P. vivax, was recently expressed in Escherichia coli and its immunogenicity was assessed in mice and Aotus monkeys. These studies indicated high immunogenicity in both animal models and the elicited antibodies displayed significant and reproducible transmission-blocking activity in ex vivo P. vivax membrane-feeding assays (MFA) [antigens . These ptructure , 8. In Petocytes  and is retocytes . In addietocytes , 11 thatetocytes –14. It ietocytes . Pvs48/4ys (MFA) .pvs48/45 among P. vivax parasite populations in malaria-endemic locations [pvs48/45 sequences corresponding to parasites from eight countries from around the world were analysed for gene diversity to assess probable protein changes that could influence the immunogenicity and its vaccine potential.Genetic diversity could generate antigenic polymorphisms, which in turn could induce changes in critical epitopes and hamper vaccine efficacy. Successful development of an effective transmission-blocking vaccine is likely dependent on an assessment of the degree of genetic diversity in ocations . Althougocations , 18, knoBlood samples used in this study were obtained from studies approved by the Institutional Review Board (IRB) of the Malaria Vaccine and Drug Development Center (MVDC) under the codes CIV-01-042009, CIV 08-102010 and CIV 009. Samples from volunteers were not linked to the identity of the donor. Written informed consent was obtained from each volunteer at enrolment. All volunteers were adults over 18 years of age.pvs48/45 was studied among 60 P. vivax isolates from endemic regions of Brazil, Colombia and Honduras in Latin American (LA). The geographical origin of each of the LA isolates analysed in this study is listed in Table P. vivax isolates (n = 13) were obtained from patients attending a tertiary healthcare centre in Manaus (Amazonas State) [Plasmodium vivax Hybrid Selection initiative, Broad Institute [The genetic diversity of s State) ; Colombis State) ; Honduras State) , North Ks State) , India as State) , Thailans State) , Mexico,nstitute , 23, andP. vivax and rule out P. falciparum co-infection as previously described [Parasite genomic DNA was extracted from filter-paper blood spots using 10 % Chelex 100  and from EDTA-blood samples using the salting-out method , and werescribed .pvs48/45 of Salvador I strain (PlasmoDB Id: PVX_083235). Amplification was performed in 25 μL reactions containing 2–4 μL genomic DNA, 500 nM of primers Pvs48/45F and Pvs48/45R, and 0.5 units of HotStar HiFidelity Polymerase . Thermal cycling conditions were as follows: initial denaturation for 5 min at 94 °C, followed by 39 cycles of: 94 °C for 30 s, 55 °C for 1 min, 72 °C for 2 min, and a final extension step at 72 °C for 10 min. Amplification products were analysed by gel electrophoresis in 1.2 % agarose gels and purified using the High Pure PCR product purification kit, according to the manufacturer’s recommendations . Purified PCR products were sequenced using the Big Dye Terminator Kit v3.1  in an ABI-PRISM Avant 3100 Automatic Genetic Analyzer  using primers Pvs48/45F and Pvs48/45R. Each purified product was sequenced in both directions by a minimum of two independent sequencing reactions until quality base calling value scores >20 (predictive sequencing error rate per base of 1.00 %) were obtained [Pvs48/45F (5′-GGAATAATTTCGACCACTC-3′) and Pvs 48/45R (5′- TCAGAAGTACAACAGGAG-3′) primers were designed to amplify the Pvs48/45 gene coding region (CDS annotation 1357 bp) from the obtained , as asseP. vivaxpvs48/45 sequence reported in PlasmoDB (accession No. PVX_083235) using the reference assembler algorithm in Geneious 4.8.4 [K), number of synonymous (dS) and non-synonymous (dN) substitutions with DnaSP 5.10 package using the Jukes-Cantor model. DNA polymorphism was analysed with a sliding window of 100 bases and a step of 25 bases for a haploid genome [All sequences were assembled against the Sal I us 4.8.4  and corrd genome . To deted genome  using thd genome  with thed genome . Null hyPlasmodium knowlesi pvs48/45 as an outgroup were performed on DNASP 5.10 to test for the neutral theory of evolution. Haplotypes reported in this study were compared to 132 pvs48/45 sequences reported in GenBank for South Korea [Pfs48/45 3D7 amino acid sequence (PFD3D7_1346700) to ensure the localization of the single nucleotide polymorphisms (SNPs) in the Cys-rich domains (CRD) I, II, and III were epitopes I, II, III and V as previously reported [Tajima’s D test and Fu and Li’s D and F tests using th Korea , North Kth Korea , India ath Korea , and refreported . PhylogeIn order to determine the presence of linear B cell epitopes in the Pvs48/45 protein sequence and the possible influence of the SNP in the predicted epitopes, an in silico analysis of the entire sequence was performed. Epitopes were predicted based on hydrophilicity, accessibility, polarity, flexibility estimation, combined with secondary structure analysis (Kolaskar-Tongaonkar antigenicity prediction methods) .Pf12 from P. falciparum [Toxoplasma gondii (Protein Data Bank IDs: 2YMO:A and 1KZQ:A) were used as templates. Sequence alignments were obtained by using a HHpred server [Structural models of Pvs48/45 domains were built by using homology-based modelling. The known 3D structures of homologous proteins lciparum  and the d server , Clustald server  and manud server . ResultiP. vivax infections. PCR amplification of the Pvs48/45 gene-coding region showed a single band of ~1.3 kb in all samples, indicating an absence of size polymorphism. Comparison of the nucleotide sequences against the pvs48/45 reference sequence  showed that SNPs occurred at 15 positions. Nine of these mutations resulted in amino acid changes (non-synonymous substitutions) at positions E35K, Y196H, H211N, K250N, D335Y, E353Q, A376T, K390T, and K418R , in which three resulted from trimorphic polymorphisms, two from dimorphic polymorphisms, and one from a single polymorphism. None of these substitutions was common in all isolates. The amino acid substitution E353Q was found in haplotypes I–IV. The K250N substitution was found in haplotypes I, II, IV, V, and VII, while the H211N amino acid substitution was found in haplotypes I and V. Amino acid substitutions A108T, P264H, N324S, and K370N were only found in haplotypes IV, VI, VII, and V, respectively  with the nine polymorphic positions reported for South Korea, North Korea, India and Indonesia showed a set of amino acid substitutions exclusively present among LA isolates. This set contained the substitution E353Q, which is the most frequent among LA isolates (41.9 %), together with the less frequent substitutions T108A (2.3 %), P264H (2.3 %), N324S (2.3 %), and K370N (2.3 %). On the contrary, D335Y, which is highly conserved among Asian isolates (95.5 %), was not detected among LA isolates. Amino acid variations H211N and K250N are found in both Asian and LA isolates. These two substitutions, highly conserved among Asian isolates  were only found in 18.6 and 39.5 % of P. vivax LA isolates, respectively. Other substitutions previously reported for Asian isolates [Pv48/45 in worldwide samples was at least one order of magnitude lower than that of known blood-stage antigen genes, such as pvmsp1, pvmsp3a and pvdbp  haplotype predominated among Brazilian (92 %), Peruvian (90 %), Mexican (87 %), and Honduran (71 %) isolates, however in Colombian isolates, it was the second-most frequent haplotype (24 %). Colombian isolates showed a major presence of haplotype I (52 %), which was less frequent in Honduran isolates (14 %), and not detected among Brazilian, Peruvian and Mexican samples. Haplotype VI, the most frequent in Myanmar and Thailand, was found only among Colombian isolates, whereas haplotype VII was only present in Colombia isolates. Colombia showed the greatest haplotype diversity (0.841 ± 0.024) with eight non-Sal I haplotypes circulating in the country.A total of 12 linear epitopes were identified in regions 1, 5, 6, and 12, displaying greater algorithmic scores predictive of putative antigenic peptides Table . A high Pvs48/45 3D model domains showed that these residues are located in the loop regions.A 3D model of Pvs48/45 was generated to understand the potential structure–function relationship of the nine polymorphic amino acids under balancing selection. SNPs were located in different regions of Pvs48/45: H211N (18.6 %) was in an intervening region between domains 1 and 2; K250N (39.5 %) was located in domain CRD-2, which is not considered a six-cys domain; and E353Q (41.9 %), A376T (50.7 %), K390T (1.1 %), and K418R (51.7 %) in domain CRD-3, which is the nearest to the trans-membrane region , CRD-2 (H211N and K250N) and CRD-3 . Pvs48/45 was more conserved in the Korean populations where nucleotide diversity varied from 0.00053  to 0.001P. falciparum in Kenyan, Thai, Indian, and Venezuelan isolates [Results are in agreement with previous observations that transmission-blocking vaccine candidate antigens Pvs25, Pvs28, Pvs48/45, and PvWARP showed limited sequence polymorphisms . More imisolates , 40. HowPvs48/45 Cys domains are important for proper conformation of immune epitopes , 46.Substitutions H211N and K250N are predicted to be located in loop regions , which cP. vivax sexual antigens the exposure to the immune system could be longer than in P. falciparum due to the early appearance of P. vivax gametocytes in circulation [Although parasite antigen diversity has been explained in part by immune pressure on the parasite –43, it dculation . HoweverP. vivax malaria it has been observed that gametocytogenesis occurs earlier than in P. falciparum and remains active even in asymptomatic carriers [Transmission-blocking vaccines have been considered a promising strategy/tool for malaria control/elimination. In carriers . Consequcarriers .pvs48/45 with noted geographical clustering among Asian and American isolates. The low genetic diversity of the protein does not influence the predicted antigenicity or protein structure and, therefore, supports its further development as a transmission-blocking vaccine candidate with widespread global potential.There is limited sequence polymorphism in


Plasmodium falciparum until resistance arose. One approach to reversing resistance is the inhibition of chloroquine efflux from its site of action, the parasite digestive vacuole. Chloroquine accumulation studies have traditionally relied on radiolabelled chloroquine, which poses several challenges. There is a need for development of a safe and biologically relevant substitute. We report here a commercially-available green fluorescent chloroquine-BODIPY conjugate, LynxTag-CQGREEN, as a proxy for chloroquine accumulation. This compound localized to the digestive vacuole of the parasite as observed under confocal microscopy, and inhibited growth of chloroquine-sensitive strain 3D7 more extensively than in the resistant strains 7G8 and K1. Microplate reader measurements indicated suppression of LynxTag-CQGREEN efflux after pretreatment of parasites with known reversal agents. Microsomes carrying either sensitive- or resistant-type PfCRT were assayed for uptake; resistant-type PfCRT exhibited increased accumulation of LynxTag-CQGREEN, which was suppressed by pretreatment with known chemosensitizers. Eight laboratory strains and twelve clinical isolates were sequenced for PfCRT and Pgh1 haplotypes previously reported to contribute to drug resistance, and pfmdr1 copy number and chloroquine IC50s were determined. These data were compared with LynxTag-CQGREEN uptake/fluorescence by multiple linear regression to identify genetic correlates of uptake. Uptake of the compound correlated with the logIC50 of chloroquine and, more weakly, a mutation in Pgh1, F1226Y.Chloroquine was a cheap, extremely effective drug against  Plasmodium falciparum. Chloroquine (CQ), once a spectacularly successful antimalarial drug, was first discovered by the German chemist Johann Andersag but was mistakenly thought to be too toxic for therapeutic purposes, an incident which became known as “the resochin error” (resochin being the name given to the compound by Andersag) pfcrt  gene, which codes for a transporter situated on the membrane of the parasite digestive vacuole (DV).Despite years of intense global effort to eradicate it, malaria is still one of the deadliest infectious diseases, killing more than 600 000 people in 2010 alone s-indacene (BODIPY) During parasite development in the intraerythrocytic cycle, haemoglobin is digested in the DV and the toxic heme moiety is released, which the parasite crystalizes into non-toxic hemozoin P. falciparum. We therefore present here the characterization of a commercially-available BODIPY-CQ conjugate, LynxTag-CQGREEN, in several laboratory strains and clinical isolates.BODIPY derivatives typically exhibit strong fluorescence and are relatively inert in biological conditions P. falciparum laboratory strains 3D7 (MRA-102), K1 (MRA-159), 7G8 (MRA-154), HB3 (MRA-155), CS2 (MRA-96), T9-94 (MRA-153), and Dd2 (MRA-156) were obtained from MR4, ATCC Manassas Virginia. Strain T9/96 was obtained from The European Malaria Reagent Repository. A further twelve clinical isolates were collected from the Mae Sot district, Tak Province, in northwest Thailand at the Shoklo Malaria Research Unit; these isolates are prefixed ‘SMRU’. Parasites were continuously cultured in complete malaria culture media (MCM) consisting of RPMI 1640 (Life Technologies) supplemented with 0.5% (w/v) Albumax I (Invitrogen), 0.005% (w/v) hypoxanthine, 0.03% (w/v) L-glutamate, 0.25% (w/v) gentamycin, with human erythrocytes at 2.5% haematocrit. Cultures were gassed with a mixture of 3% CO2, 4% O2 and 93% N2 and incubated at 37°C. Synchronization of parasite cultures was performed by resuspending erythrocytes in 5% (w/v) D-sorbitol and incubating at 37°C for 10 min, after which the erythrocytes were washed twice, resuspended in MCM and returned to culture conditions. Thin Giemsa smears were made before each experiment to determine parasitemia and parasite stage.GREEN  was dissolved in DMSO to the same concentration. All compounds were stored at −20°C and protected from light. For microsome uptake assays, methiothepin mesylate salt, metergoline, loperamide hydrochloride, octoclothepin maleate salt, mibefradil dihydrochloride hydrate, L703,606 oxalate salt hydrate, and chlorprothixene hydrochloride  were dissolved in DMSO to 10 mM and stored at 4°C. Verapamil hydrochloride, adenosine triphosphate (ATP), and CQ diphosphate  were dissolved in water to 7.5 mM, 50 mM and 0.1 M respectively and stored at −20°C. Tritiated CQ  was diluted in water to 5.32 µM and stored at −20°C; specific activity was 4.7 Ci/mmol.For work involving parasites, chlorpheniramine maleate salt, chlorpromazine hydrochloride, desipramine hydrochloride, promethazine hydrochloride, verapamil hydrochloride and CQ diphosphate  were dissolved in PBS to a working concentration of 1 mM. LynxTag-CQGREEN at a range of concentrations for 48 h in 96-well flat-bottomed plates at culture conditions. Following this, cells were stained with 1 µg/ml of Hoechst 33342 (Invitrogen) for 30 min at 37°C, washed twice and resuspended in PBS. Parasitemia was then assessed with the CyAn ADP flow cytometer (Beckman Coulter). IC50s were determined by plotting the measurements in Graphpad Prism 5 using a variable slope logistic curve.Synchronized ring-stage cultures at 1–2% parasitemia, 1.25% haematocrit were incubated with either CQ or CQGREEN for 2 h at 2 µM in a 96-well plate format. Erythrocytes were then washed twice and stained with Hoechst 33342 as previously. Wet mounts of stained parasites were visualized under ×100 magnification with the Fluoview FV1000 confocal microscope (Olympus). Hoechst and CQGREEN were excited at 405 nm and 488 nm with emissions captured at 430–470 nm and 505–525 nm respectively.200 µl cultures of 3D7 at 3% parasitemia, 1.25% haematocrit were incubated with CQGREEN to 2.5% haematocrit in a 96-well plate format. The parasites were then incubated for 2 h at culture conditions, after which they were washed twice and resuspended in PBS. Cells were allowed to settle in a Nunc F96 MicroWell black non-treated polystyrene plate (Thermo Scientific) for 1 h. Fluorescence was then measured with the Infinite M200 microplate reader (Tecan) with excitation and emission wavelengths of 488 nm and 520 nm respectively. K1 chemoreversal assays were performed by pretreatment with 10 µM of the reversal agents for 30 min prior to the addition of CQGREEN.Synchronized trophozoite-stage cultures at 3–5% parasitemia were resuspended in 200 µl of MCM with 2 µM of CQP. falciparum strains Dd2 or 3D7 were expressed in Pichia pastoris KM71 and microsomes harvested as described previously PfCRT originating from GREEN uptake by the microsomes, total or non-specific uptake was measured. Non-specific uptake was determined by pretreating microsomes with unlabelled CQ at 1000 times of the concentration of CQGREEN used, for 15 min at 37°C, before adding CQGREEN; total uptake was determined without the pretreatment. Reactions were carried out in accumulation buffer . Following this, microsomes were washed twice in accumulation buffer, then lysed in lysis buffer  on ice for at least one hour. Unlabelled CQ was excluded from the washing step as the addition of CQ was observed to cause displacement of CQGREEN from the microsomes, possibly due to the higher affinity of CQ for PfCRT. Measurement of the fluorescence intensity was performed using the FLUOstar Galaxy microplate reader (BMG Labtech) with excitation and emission wavelengths of 480 nm and 520 nm respectively. For each experiment, measurements were made in triplicate and the mean calculated. Specific uptake was then calculated by subtracting non-specific uptake from total uptake. Non-linear regression analysis with the Michaelis-Menten model (GraphPad Prism 5) was then applied to obtain the Vmax and Km of specific uptake.In order to assess the Michaelis-Menten kinetics of CQGREEN at 37°C for 15 min. For uptake inhibition assays, microsomes were pre-incubated with chemoreversal compounds GREEN. All data presented are specific uptake based on the calculations stated above.Unless stated otherwise, microsomes were incubated with 15 µM CQ3H-CQ uptake was measured as described previously 3H-CQ at 308 nM for 5 min, after which 200-fold unlabelled CQ was added to stop the reaction. Microsomes were then precipitated by the addition of polyethylene glycol (PEG) 8000 and washed twice with accumulation buffer containing 200-fold unlabelled CQ to remove excess 3H-CQ. Microsomes were then resuspended in scintillation buffer and agitated overnight. Radioactivity was measured using the LS 5600 Scintillation Counter (Beckman). All data presented are specific uptake.pfmdr1 polymorphisms, parasite DNA from in vitro cultures was extracted with the QIAamp DNA Mini kit (Qiagen) as per the manufacturer's instructions. For pfcrt polymorphisms, total RNA was extracted with the RNeasy Mini Kit (Qiagen) and reverse transcription performed with SuperScript III (Invitrogen) as per manufacturers' instructions. Polymerase chain reaction (PCR) mixtures were made with 200 µM of each dNTP, 0.5 µM forward primer, 0.5 µM reverse primer, 0.02 U/µl Phusion DNA polymerase (Thermo Scientific), 6 µl of 5× Phusion HF buffer, and 1 µl of genomic DNA or cDNA to a total reaction volume of 30 µl. Thermocycler parameters were as follows: 98°C for 30 s, followed by 35 cycles of 98°C for 10 s, 60°C for 30 s, and 72°C for 1 min. Primers used for pfmdr1 sequencing were 5′- ATGGGTAAAGAGCAGAAAGA and 5′- TCCACAATAACTTGCAACAGT, or 5′- GTCAAGCGGAGTTTTTGC and 5′- TATTCTCTGTTTTTGTCCAC. Pfcrt-specific primers were 5′- GACGAGCGTTATAGAGAAT and 5′- CTTCGGAATCTTCATTTTCT. PCR products were purified with the QIAquick PCR purification kit as per manufacturer's instructions. Purified PCR products were sequenced by a commercial vendor . Copy number of pfmdr1 was assessed by real-time PCR as previously reported 2, 300 nM dNTPs, 300 nM each of forward and reverse primers, and 100 nM of the probe. Thermocycler parameters were 95°C for 10 min, then 40 cycles of 95°C for 15 s and 60°C for 1 min. Forward and reverse primers used were 5′- TGCATCTATAAAACGATCAGACAAA and 5′- TCGTGTGTTCCATGTGACTGT respectively, and TaqMan probe was 5′- 6FAM-TTTAATAACCCTGATCGAAATGGAACCTTTG-TAMRA. A reference gene, β-tubulin, was also included; primers were 5′- TGATGTGCGCAAGTGATCC and 5′- TCCTTTGTGGACATTCTTCCTC, while the probe was 5′- VIC-TAGCACATGCCGTTAAATATCTTCCATGTCT-TAMRA. The threshold cycle (Ct) was analysed by the comparative Ct method, based on DNA amplification efficiencies of the pfmdr1 and β-tubulin genes. Pfmdr1 copy number was calculated according to the following formula: ΔCt  =  CtR - CtG, where CtR is the reference β-tubulin Ct, and CtG is that of pfmdr1. Each TaqMan run included three reference DNA samples from clones 3D7, K1, and Dd2 having pfmdr1 copy numbers of 1, 1, and 3 respectively.To assess 50s and with dummy coded values for the respective amino acid residues.All statistical analyses were performed with SPSS 21. Chemoreversal assays were assessed with Student's t test, 2-tailed. Multiple linear regression was performed with the stepwise method, using the log of ICin vitro malaria culture was approved by the Institutional Review Board  of the National University of Singapore (NUS). All participants provided written informed consent. The clinical isolates used were obtained under ethical guidelines in the approved protocol: OXTREC Reference Number 29–09 . Use of field isolates in NUS was in accordance with NUS IRB (Reference Code: 12–369E).The blood collection protocol for GREEN in the parasite. Cultures of P. falciparum 3D7 were co-stained with Hoechst dye and CQGREEN, revealing a preferential accumulation of CQGREEN in the parasite DV , uptake was close to saturated. Michaelis-Menten approximation of CQGREEN uptake kinetics in Dd2 microsomes   it is differentially recognized by CQ-resistant versus CQ-sensitive PfCRT, (2) its uptake by PfCRT is ATP-dependent, and (3) its uptake by CQ-resistant PfCRT is verapamil-reversible. Others have shown that extensive CQ side-chain modifications can render the CQ analogues not transportable by PfCRT and abolish their verapamil sensitivity GREEN still allows CQGREEN to be differentially recognized by resistant versus sensitive PfCRT and these transport activities are still sensitive to verapamil. To demonstrate the usefulness of CQGREEN in screening for PfCRT inhibitors, we found that CQGREEN uptake by Dd2 PfCRT microsomes can be inhibited by mibefradil, a novel potent chemosensitizer GREEN uptake by a panel of known chemoreversal agents was compared to that using 3H-CQ , showing moderately good agreement between CQGREEN and 3H-CQ uptake (R2 = 0.766). However, 3H-CQ uptake was roughly 100 times greater than that of CQGREEN.To test if CQcrosomes  yields aCRT/min) . PfCRT-mcrosomes . VerapamD7 PfCRT . These rng 3H-CQ . Mean uppfcrt and pfmdr1 respectively. Residues examined were 72, 74, 75, 76, 220, 271, 326, 356, and 371 for PfCRT, and 86, 184, 1034, 1042, 1226, and 1246 for Pgh1. These residues were chosen for analysis as they were previously implicated in modulating multidrug resistance as well as resistance against CQ pfmdr1 was also determined for each strain and isolate. All clinical isolates showed Dd2-type PfCRT mutations, whereas Pgh1 mutations and pfmdr1 copy numbers were more varied . None of the other mutations was significantly correlated with CQGREEN uptake. Separating the data set to two subpopulations improved the R2, to 0.72 in the Pgh1 1226F group and 0.676 in the Pgh1 1226Y group (GREEN uptake, given that it is also correlated with resistance to artemisinin, mefloquine and lumefantrine GREEN uptake could range as high as that of the CQ-susceptible strains (GREEN uptake could be pfmrp, which has been proposed to efflux drugs across the parasite plasma membrane into the parasitophorous vacuolar lumen GREEN fluorescence but sequester the drug from its site of action. Knock-out mutants of this gene exhibit increased susceptibility to CQ, quinine, artemisinin, piperaquine, and primaquine All strains and isolates were assayed for CQ of 0.53 . However6Y group . It is t strains  while stGREEN is a commercially available fluorescent CQ analog that interacts with the parasite in a similar fashion to CQ. CQGREEN presents some advantages over traditional radiolabelled CQ in uptake studies: it is safer to handle as it is not radioactive, and its fluorescence properties allows it to be monitored by common fluorescence equipment. Using a defined microsomal platform, we showed that CQGREEN interacts with PfCRT in a manner similar to CQ. Its use as a predictor of CQ susceptibility is enhanced if residue 1226 of Pgh1 is known. Unlike a typical reinvasion assay which may require 48 h or more, the use of CQGREEN allows for measurement of CQ susceptibility within several hours. We believe that CQGREEN could be a valuable tool in future drug discovery projects or used in the identification of factors involved in drug resistance.CQ


Quetiapine fumarate is an antipsychotic drug with poor oral bioavailability (9%) due to first-pass metabolism. Present work is an attempt to improve oral bioavailability of quetiapine fumarate by incorporating in solid lipid nanoparticles (SLN). Six quetiapine fumarate SLN formulations were developed using three different lipids by hot homogenisation followed by ultrasonication. The drug excipient compatibility was studied by differential scanning calorimetry (DSC). Stable quetiapine fumarate SLNs having a mean particle size of 200–250 nm with entrapment efficiency varying in between 80% and 92% were developed. The physical stability of optimized formulation F3 was checked at room temperature for 2 months. Comparative bioavailability studies were conducted in male Wistar rats after oral administration of quetiapine fumarate suspension and SLN formulation. The relative bioavailability of quetiapine fumarate from optimized SLN preparation was increased by 3.71 times when compared with the reference quetiapine fumarate suspension. The obtained results are indicative of SLNs as potential lipid carriers for improving the bioavailability of quetiapine fumarate by minimizing first-pass metabolism. Quetiapine fumarate is an antipsychotic drug with plasma half life of 6 h and poor oral bioavailability (9%) due to extensive first-pass metabolism . PossiblSolid lipid nanoparticles (SLNs) are an alternative nanoparticulate carrier system to polymeric nanoparticles, liposomes, and o/w emulsions –8. AqueoThere are very few reports in literature describing the use of SLNs for bypassing first-pass metabolism. When all-trans-retinoic acid was loaded into SLNs, the oral bioavailability in rats was increased four- to five-fold compared with that of suspension . The oraIn the present study, the quetiapine fumarate loaded SLNs were prepared using glyceryl trimyristate, glyceryl tristearate, and glyceryl monostearate as lipids by hot homogenization followed by ultrasonication method. The prepared SLNs were characterized, and an optimized SLN formulation was used to assess the oral bioavailability improvement of quetiapine fumarate. Quetiapine fumarate was obtained as a gift sample from Aurobindo Labs, Hyderabad. Dynasan 114 (glyceryl trimyristate), Dynasan 118 (glyceryl tristearate) and Imwitor 900P (glyceryl monostearate) , egg lecithin , Poloxamer-188 , chloroform , methanol , dialysis membrane  were purchased from the local market. All the other reagents used were of analytical grade.Quetiapine fumarate loaded SLNs were prepared by hot homogenization followed by the ultrasonication . The comDSC scan was performed by Mettler-Toledo DSC 821e  instrument. DSC scans were recorded for the entire drug and lipid combinations at a heating rate of 10°C/min in temperature range of 50–250°C. μL of the prepared SLN dispersion was diluted to 5 mL with double distilled water and analyzed with zetasizer.The size, PDI, and ZP of quetiapine fumarate SLNs were measured using a Malvern Zetasizer . About 100 Entrapment efficiency was determined by measuring the concentration of free drug (unentrapped) in aqueous medium as reported . The aquμL of the formulation was dissolved in 0.9 mL of chloroform and methanol mixture (1 : 1), and then further dilutions were made with mobile phase. The diluted samples were estimated by HPLC for the amount of quetiapine fumarate present [About 100  present .In vitro release studies were performed using dialysis method. Dialysis membrane  having pore size 2.4 nm and molecular weight cut-off between 12,000–14,000 was used for the release studies. Dialysis membrane was soaked overnight in double distilled water prior to the release studies. Hydrochloric acid (0.1 N) and phosphate buffer pH 6.8 were used as release media. The experimental unit had donor and receptor compartments. Donor compartment consisted of a boiling tube which was cut open at one end and tied with dialysis membrane at the other end into which 1 mL of SLN dispersion was taken for release study. Receptor compartment consisted of a 250 mL beaker which was filled with 100 mL release medium, and the temperature was maintained at 37 ± 0.5°C. At 0.25-, 0.5-, 1-, 2-, 3-, 4-, 6-, 8-, 10-, 12-, and 24-hour time points, 1 mL samples were withdrawn from receiver compartment and replenished with the same volume of release medium. The collected samples were suitably diluted and analyzed by UV-visible spectrophotometer  at 209 nm [Quetiapine fumarate loaded solid lipid nanoparticles (optimized formulation F3) were stored at room temperature (25°C/60 ± 5% RH) and refrigerated temperature (4°C) for 60 days, and average size, zeta potential, poly dispersity index and EE were determined in triplicate.A single dose bioavailability study was designed in male Wistar rats under fasting conditions. The oral bioavailabilities of the optimized SLN formulation (F3) and suspension (F7) were estimated by conducting bioavailability studies in male Wistar rats with oral dose of 10 mg/kg body weight. All experimental procedures were reviewed and approved by the institutional animal ethical committee of University College of Pharmaceutical Sciences, Kakatiya University . Male Wistar rats weighing 200–250 g were taken for study . Blood samples were withdrawn by retro-orbital venous plexus puncture at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, and 24 h after dose. About 1.5 mL of blood samples were withdrawn in eppendorf tubes and centrifuged at 3000 rpm for 30 min. The plasma was transferred to another eppendorf tube and stored at −20°C until analysis.μL of serum, 10 μg/mL of felodipine  was added, and then alkalinization was achieved by the addition of 0.1 mL of NaOH (0.1 M), and the tubes were shaken for 1 min. Then about 5 mL of ether was added and vortexed for 5 min. After vortex mixing for 5 min, the mixtures were centrifuged at 3000 rpm for 6 min at room temperature. Of the upper layer, 4 mL was carefully aspirated, and the remainder was extracted once again with 5 mL of ether. Of the upper layer, 4 mL was collected together with the former. The ether was evaporated at room temperature. The residue was reconstituted with 100 μL of methanol, and reconstituted samples were analyzed using HPLC [To 100 ing HPLC . Mobile phase: acetonitrile: 0.02 M phosphate buffer pH 5.5 (65 : 35),  flow rate: 1 mL/min,  μm particle size), column: Lichrospher C-18  of quetiapine fumarate SLNs to the oral suspension was calculated as follows:In the present study, quetiapine fumarate loaded SLNs were prepared by hot homogenisation followed by ultrasonication method using three lipids, each at two different concentrations. The selection and utility of lipids and method of preparation were based on the earlier reports. Dynasan-114, Dynasan-118, and Imwitor-900P were known to produce SLNs. Due to the reports and previous observations, these lipids were tried in our studies as excipients. Egg lecithin and poloxamer were known as surfactants to get the dispersions of SLNs. At first instance, hot homogenization followed by ultrasonication method was tried, which yielded better SLNs. Inspite of availability of other methods of preparation, we used this method only as this resulted in consistent production of smaller size nanoparticles (<250 nm) with narrow size distribution and good entrapment efficiency.DSC thermograms of pure drug and physical mixtures of drug and different lipids are shown in Drug-related peaks are seen at right side of thermograms and lipid-related peaks are on left side of the All the prepared samples were analyzed in order to determine their particle size distribution, zeta potential, and PDI values. The results are represented in Entrapment efficiency is an important parameter for characterizing solid lipid nanoparticles. All the formulations were analyzed for entrapment efficiency by HPLC, and the results are shown in All the formulations were analyzed for total drug content by HPLC, and the results are represented in Formulations containing Dynasan-114 (F1 and F2), Dynasan-118 (F3 and F4) and Imwitor-900P (F5 and F6) showed cumulative drug release ranging from 55.24% (F2) to 70.89% (F1), from 58.40% (F4) to 71.24% (F3) and from 55.55% (F6) to 65.32% (F5) respectively in 0.1 N Hydrochloric acid  during 2Formulations containing Dynasan-114 (F1 and F2) showed drug release ranging from 30.20% (F2) to 40.63% (F1) in 6.8 pH phosphate buffer. Formulations containing Dynasan-118 (F3 and F4) showed drug release ranging from 40.26% (F4) to 54.57% (F3) in 6.8 pH phosphate buffer .in vitro release of drug from SLN is less in phosphate buffer pH 6.8 when compared to 0.1 N HCl. Quetiapine fumarate solubility is more in acid medium than in phosphate buffer pH 6.8. This increase in solubility is probably responsible for increase in the cumulative drug release in acid medium. Further, increased lipid content reduced the cumulative release which is in consistent with the earlier reports. The lipid content increases the packing density of lipid molecules in given space; as a result release is reduced. For further studies, based on the particle size, PDI, zeta potential, and in vitro release, F3 was considered as optimized formulation.Formulations containing Imwitor-900P (F5 and F6) showed drug release ranging from 36.63% (F6) to 40.80% (F5) in 6.8 pH phosphate buffer. When compared, the cumulative release in 6.8 pH phosphate buffer was less than that of 0.1 N HCl. Formulation (F3) containing Dynasan-118 showed maximum release of 54.47% in 6.8 pH phosphate buffer in 24 hours. In general, the t-test was applied. Only size of SLNs was changed, but no significant difference was noticed in EE, PDI, and ZP of the SLNs during the storage period. Thus, the optimized SLN preparation was stable for two-months period at RT and at 4°C.Quetiapine fumarate loaded solid lipid nanoparticles were stored at room temperature (25°C/60 ± 5%RH) and refrigerated temperature (4°C) for 60 days, and average size, zeta potential, poly dispersity index, and entrapment efficiency (EE) were determined. Stability studies were conducted for optimized formulation (F3) which showed better size, PDI, zeta potential, and EE. The number of samples estimated was in triplicate. The results are shown in μg/mL, whereas in the case of suspension, the peak plasma concentration is 0.107 ± 0.004 μg/mL. The tmax is same for both suspension and SLN. The plasma concentration-time profiles of suspension and SLN formulation are shown in The drug in the serum samples was estimated by using HPLC method . VariousCmax and AUC0–24 for suspension (F7) were lower than that of the optimized SLN formulation (F3). The pharmacokinetic parameters were subjected to statistical analysis using unpaired t-test with help of Graphpad Prism software version 5.0, 2007. Significant difference was noticed in Cmax, AUC, and t1/2 values for F3 when compared to the suspension (F7). Further, the relative bioavailability of the SLN formulation F3 was found to be 3.71 times that of suspension F7. The oral bioavailability of quetiapine fumarate from the formulation F3 was higher than that of suspension F7 formulation. This enhancement might be due to lymphatic transport of drug from SLN formulation and avoiding first-pass metabolism of drug. From above results, it was found that in vivo bioavailability studies 3.71 times of relative bioavailability improvement was found when compared to reference suspension. Thus, quetiapine fumarate when formulated as SLN could improve the oral bioavailability.Poorly bioavailable quetiapine fumarate was formulated as SLN using three different lipids after checking the compatibility by DSC studies. The SLN preparation with Dynasan 118 was optimized based on the particle size, PDI, zeta potential, entrapment efficiency, and drug release characteristics. During


More than half of intravenous drug users (IDUs) in China suffer from the Hepatitis C virus (HCV). The virus is also more prevalent in non-injection drug users (NIDUs) than in the general population. However, not much is known about HCV subtype distribution in these populations.Our research team conducted a cross-sectional study in four provinces in China. We sampled 825 IDUs and 244 NIDUs , genotyped each DU’s virus, and performed a phylogenetic analysis to differentiate HCV subtypes.Nucleic acid testing (NAT) determined that 82% percent (952/1162) of samples were HCV positive; we subtyped 90% (859/952) of these. We found multiple HCV subtypes: 3b , 3a , 6a , 1b , 6n , 1a , and 2a . An analysis of subtype distributions adjusted for province found statistically significant differences between HCV subtypes in IDUs and NIDUs.HCV subtypes 3b, 3a, 6a, and 1b were the most common in our study, together accounting for 89% of infections. The subtype distribution differences we found between IDUs and NIDUs suggested that sharing syringes was not the most likely pathway for HCV transmission in NIDUs. However, further studies are needed to elucidate how NIDUs were infected. A meta-analysis of studies from 25 countries estimated that 60–80% of intravenous drug users (IDUs) were infected with the Hepatitis C virus (HCV).  In ChinaHCV treatment can be effective in reducing risk for HCV-related liver diseases and HCV secondary transmission.  However,To describe HCV subtype distribution among DUs in China, we conducted a cross-sectional study in four provinces. Between 2008 and 2011, we sampled 1,162 HCV-infected DUs from the national networks of methadone clinics and the HIV sentinel surveillance clinics in the Chinese provinces of Guizhou, Hebei, Jiangsu, and Shanxi. Two rounds of HCV-antibody enzyme-linked immunsorbent assays (ELISA) were used to determine HCV serostatus. All our study participants tested positive in both rounds. The national clinics de-identified participants’ demographic data and sent both the data and their blood samples to the Center for Disease Control (CDC) in China. The de-identified dataset still included demographic and behavioral information. Our study was approved by the Vanderbilt University School of Medicine IRB (number: 131253).Roche Ltd, Basel, Switzerland). The nucleic acid test (NAT) to detect the virus used a nested reverse transcription polymerase chain reaction (RT-PCR) process. First, we amplified a 170bp fragment in the 5’-untranslated region (5’-UTR). The primers we used for this process have been previously published  as the reagent mixture. It contained 5μL RNA extraction, 17μL DEPC water, 25μL Mix , 1μL mixture of enzymes , 1μL sense primer (5’-UTR-IS), and 1μL antisense primer (5’-UTR-IAS). We ran this first RT-PCR round for 30 min. at 50°C, 2 min. at 94°C, followed by 30 cycles of 30 sec. at 94°C, 30 sec. at 55°C, and 1 min. at 72°C. We allowed 7 min. at 72°C for elongation, and then stored the PCR product at 4°C. Third, we ran another nested-PCR step using 2×Taq Master Mix  as the reaction mixture. It contained 2μL PCR products from RT-PCR, 21μL DEPC water, 25μL Mix , 1μL sense primer (5’-UTR-NS), and 1μL antisense primer (5’-UTR-NAS). We ran this round for 2 min. at 94°C, followed by 34 cycles of 30 sec. at 94°C, 30 sec. at 55°C, and 1 min. at 72°C. We used the same elongation and storage protocol as we did in the first step.At the CDC, we used participants’ blood samples to determine their HCV subtypes. We extracted HCV RNA using the MagNA Pure LC automated platform , and then used gel electrophoresis to detect the target DNA fragments.For the RNA samples that tested positive for HCV, we amplified a 350bp fragment in the NS5B region. We then applied a nested RT-PCR procedure similar to the NAT procedure, except using different primers . We usedhttps://hcv.lanl.gov/content/sequence/HCV/ToolsOutline.html). Finally, we ran Mega 4.0 to conduct the phylogenetic analyses. Subtype information was read from the resulting phylogenetic trees (using the neighbor-joining method with the Kimura-2-parameter).We used ChromasPro 1.5 to trim and assemble sequences. Then we used BioEdit 4.0 to perform multiple alignments against the NS5B reference sequences from the HCV Sequence Database .We compared HCV subtype distributions among demographic variables using a chi-squared test. To control for confounding effects by region, we analyzed the subgroups on a per-province basis. We conducted all analyses using Statistical Analysis System 9.3 . Most participants in our study population (77.2%) were IDUs.The median age in our study population was 38 years . Most participants were of Han ethnicity, male, single, and had less than an 8In this study, 952 (81.9%) DUs tested positive for HCV RNA, and we successfully subtyped 859 of those 952 (90.2%). The proportion of NAT positive and subtyping rate differed by province and sample source . For exaOur study population was on average older than those in other studies, but the ratio of males to females was similar (3.5:1). ,25 PreviThe main HCV subtypes in our DUs were 3b, 3a, 6a, and 1b, which was concordant with other studies. ,26 By coet al. found frequencies of HCV subtypes to be similar between IDUs and NIDUs. In our study, HCV subtype 1b, 3b, and 6a were the main strains in NIDUs, whereas 3a and 3b were the most common in IDUs. Our finding was biologically plausible because certain HCV subtypes are mainly transmitted by IDUs through sharing of needles, while true NIDUs must be infected via a different pathway, such as sexual transmission. We say “true NIDUs” because we observed that a small proportion of HCV sequences from IDUs and NIDUs clustered into the same group in our phylogenetic analyses . Drug use method (IDU vs. NIDU) may also be MAR, as the majority of missing responses were from participants from Guizhou. As a result, when conducting analyses between provinces, our findings could be either inflated or attenuated. As mentioned before, response bias from DUs seems to have caused some of them to be misclassified. However, we partially offset this by studying a large sample covering four geographic areas in China. We successfully replicated the geographic distribution of HCV subtype from previous studies, and discovered that the HCV subtype distribution was different between IDUs and NIDUs. Our study provided information for future HCV transmission research among NIDUs, and was potentially useful for guiding HCV treatment in China.S1 Table(DOCX)Click here for additional data file.S1 Text(PDF)Click here for additional data file.


The purpose of this study was to investigate the mitochondrial effects exerted by physiological and supra-physiological concentrations of recombinant human growth hormone (rhGH) and recombinant insulin-like growth factor-1 (rIGF-1) under conditions of substrate saturation in peripheral blood mononuclear cells (PBMCs).m) and mitochondrial levels of highly reactive oxygen species (hROS) were subsequently analysed. This analysis was performed by flow cytometry in digitonin permeabilized cells, following treatment with saturating concentrations of various respiratory substrate combinations and the use of specific electron transport chain (ETC.) complex inhibitors, enabling control over both the sites of electron entry into the ETC. at complexes I and II and the entry of electrons from reduced carriers involved in β-oxidation at the level of ubiquinol.PBMCs from healthy male subjects were treated with either rhGH, at concentrations of 0.5, 5 and 50 μg/L, or rIGF-1 at concentrations of 100, 300 and 500 μg/L for 4 h. Mitochondrial membrane potential . Such elevated supplies of lipid-derived substrates to the mitochondria could lead to oxidative damage which would negatively impact mitochondrial function. Thesolecules . In addi of hROS . The sub of hROS , 10.2− as well as overall O2− and H2O2 cellular concentration levels. Of note, previous research from our laboratory has demonstrated that four hours in the presence of rhGH, at physiological concentrations (5–10 μg/L), was enough to significantly reduce production rates of mitochondrial O2− molecules in peripheral blood mononuclear cells (PBMCs) [2− generation via direct modulation of the Δψm.A decline in the systemic release of growth hormone (GH) and its subsequent stimulation of insulin-like growth factor-1 (IGF-1) production is a well-known effect of aging in both humans and experimental animals , 11. Ind (PBMCs) . Several (PBMCs) , 17. ArdDespite the documented antioxidant effects resulting from these hormones, at supra-physiological concentrations in-vivo GH is associated with adverse effects leading to negative implications for normal physiological functions , 19. Pat2− and H2O2 production [m in digitonin permeabilized cells. This was carried out following treatment with saturating concentrations of various respiratory substrate combinations and in the presence of specific ETC. complex inhibitors, enabling control over the sites of electron entry into the ETC. at complexes I and II, but also over the entry of electrons from reduced carriers involved in β-oxidation at the level of ubiquinol.While current studies examining the effect of rhGH and rIGF-1 on cellular oxidative status have analysed levels of Ooduction , 12, 13,oduction  we hypotoduction , 24 and oduction , 26. Flo2) were recruited to participate in the study which was approved by the Bond University Human Research Ethics Committee (BUHREC-RO1134). All subjects were given information on the nature of the study and the associated risks involved. These were explained to them prior to providing written informed consent. Exclusion criteria included smoking, the use of therapeutic, recreational or performance enhancing drugs, including anabolic steroids and rhGH up to twelve months prior to participation in the study, a history of diabetes, cardiovascular associated disease or the use of prescription medications.Ten healthy male subjects ), 3′-p-hydroxyphenyl fluorescein (HPF), and sodium pyruvate were purchased from Invitrogen . L-malic acid , sodium succinate, sodium octanoate, ammonium iron (II) sulphate, H2O2, rotenone, digitonin, sucrose, potassium phosphate monobasic (KH2PO4), magnesium chloride (MgCl2), potassium morpholinopropane sulphonate (MOPS), adenosine diphosphate (ADP), ethylenediaminetetraacetic acid (EDTA) and bovine serum albumin (BSA) were purchased from Sigma Aldrich . Phosphate buffered saline (PBS) was purchased from Kinetic . Ficoll-Paque PLUS was obtained from GE Healthcare . Finally, rhGH was purchased from Pfizer  and rIGF-1 was purchased from Biocore .Hanks balanced salt solution (HBSS), RPMI Medium 1640, carbonyl cyanide m-chlorophenylhydrazone (CCCP), 1,1′,3,3,3′,3′-hexamethylindodicarbo-cyanine iodide . Collected blood was diluted in an equal volume of sterile PBS and subsequently layered over Ficoll-Paque PLUS at a ratio of 2:1. All samples were centrifuged at 450*g for 30 mins for the separation of PBMCs from whole blood. Isolated cells were washed in HBSS and their concentration was determined using a Countess automated cell counter . Finally, cells were resuspended in RPMI 1640 cell culture medium at a concentration of 1*102.Isolated PBMCs were divided into aliquots for subsequent experimentation with cells either remaining untreated or administered rhGH  or rIGF-1 . All cell samples were subsequently incubated for 4 h at 37 °C and in the presence of 5 % COm and hROS generation under differing respiratory conditions, a method adapted from the work of Pham et al. [2PO4, 5 mM MgCl2, 1 mM EDTA, 0.1 % BSA, 1 mM ADP and 20 mM MOPS – pH adjusted to 7.4). The subsequent administration choice of substrate combinations with these cells gives rise to understanding either complex I, complex II or fatty acid mediated respiration.Following the experimental procedures and treatments, plasma membranes of these cells were permeabilized for the analysis of Δψm et al. . Sample + leading to the initiation of complex I mediated respiration. Complex II is not involved under these respiratory conditions, as malate equilibrates with fumarate at concentrations above 2 mM, inhibiting the conversion of succinate to fumarate and preventing the formation of FADH2 [Administration of the combination of pyruvate (5 mM) and malate (5 mM)  activates isocitrate dehydrogenase, α-ketoglutarate dehydrogenase complex and malate dehydrogenase, which reduce NADof FADH2 .Administration of succinate (10 mM), in the presence of rotenone (20 μM) (Succ/Rot), activates SDH, which reduces FAD leading to the initiation of complex II mediated respiration. Rotenone acts to inhibit the flow of electrons through complex I and to prevent accumulation of oxaloacetate, a potent inhibitor of SDH . RotenonAdministration of the medium chain fatty acid octanoate (10 mM) and malate (2 mM)  mediate the transfer of electrons to reducing equivalents via β-oxidation. The use of octanoate, the oxidation of which is not carnitine dependent, to assess respiratory activity in isolated mitochondria has been previously demonstrated . Additio1(5). The dye accumulates in the mitochondria as a function of membrane potential at concentrations below 100nM and fluoresces at an emission maximum of 658 nm when excited at 633 nm [Mitochondrial ∆Ψ was analysed by flow cytometry using the lipophilic cationic dye, DilCt 633 nm . The gent 633 nm . The prom were incubated at 37 °C in 5 % CO2 for 5 min in the presence of 50 μM of CCCP in order to dissipate Δψm. Positive control samples for hROS were incubated at 37 °C in 5 % CO2 for 5 min in the presence of 100 μM ammonium iron (II) sulphate and 1 mM H2O2 to induce OH(•) generation via the Fenton reaction.Negative control samples for Δψ2 for 15 min in the presence of DilC1(5) at a concentration of 10nM and HPF at a concentration of 10 μM. Following incubation, all samples were washed and resuspended in 500 μL PBS, prior to immediate analysis by flow cytometry.Aliquots from each treatment condition (1 mL) were subsequently incubated at 37 °C in 5 % COm, samples were excited at a wavelength of 633 nm with fluorescent emissions detected in channels using a bandpass filter with a range of ≥650 nm. For the analysis of hROS production, samples were excited at a wavelength of 488 nm with fluorescent emissions detected in channels using a bandpass filter with a range of 515-545 nm. For all samples analysed, a total of 10,000 events were collected while lymphocyte and monocyte populations were gated and analysed separately. All data collected was analysed using Cellquest Pro software .All samples were analysed using a FACS Calibur flow cytometer . For the analysis of ΔψA factorial multivariate analysis of variance (MANOVA) was used to determine whether significant interactions existed between respiratory conditions and hormonal treatments for each of the analysed variables, while Bonferroni’s multiple comparisons were used for post hoc analysis between individual treatment conditions . Statistical significance was determined at an alpha level of 0.05.1(5) mean channel fluorescence (AU) from samples in the presence of Pyr/Mal , Succ/Rot  and Oct/Mal  compared to untreated samples under the same respiratory conditions in the lymphocyte sub-population. Fluorescence values from Succ/Rot treated samples were significantly higher compared to Pyr/Mal  and Oct/Mal  treated samples. In addition, Oct/Mal treated samples were significantly higher compared to Pyr/Mal  treated samples  mean channel fluorescence from samples in the presence of Pyr/Mal , Succ/Rot  and Oct/Mal  compared to untreated samples under the same respiratory conditions. Fluorescence values from Succ/Rot treated samples were significantly higher compared to Pyr/Mal  treated samples but were not significantly different from Oct/Mal treated samples . Oct/Mal treated samples were also significantly higher in fluorescence compared to Pyr/Mal  treated samples  mean channel fluorescence values in either lymphocyte (P = 0.97) or monocyte (P = 0.75) sub-populations at any concentration administered. Finally, no significant interaction effect between hormonal treatment and respiratory substrate condition was observed in either lymphocyte (P = 0.99) or monocyte (P = 0.99) sub- populations.Neither GH Fig.  nor IGF-2O2 and ammonium iron (II) sulphate, HPF mean channel fluorescence values (AU) were significantly higher from samples in the presence of Pyr/Mal , Succ/Rot  and Oct/Mal  compared to untreated samples under the same respiratory conditions in the lymphocyte sub-population. Fluorescence values from Pyr/Mal treated samples were significantly higher compared to Succ/Rot  and Oct/Mal  treated samples. In addition, fluorescence values from Oct/Mal treated samples were significantly increased compared to Succ/Rot samples   Fig. .Fig. 4Hi2O2 and ammonium iron (II) sulphate also significantly increased HPF mean channel fluorescence (AU) from samples in the presence of Pyr/Mal , Succ/Rot  and Oct/Mal  compared to untreated samples under the same respiratory conditions. Fluorescence values from Pyr/Mal treated samples were significantly increased compared to Succ/Rot  and Oct/Mal  treated samples. In addition, fluorescence values from Oct/Mal treated samples were significantly increased compared to Succ/Rot samples   Fig. .P = 0.90) or monocyte (P = 0.85) sub-populations at any concentration administered. Finally, no significant interaction effect between hormonal treatment and respiratory substrate condition was observed in either lymphocyte (P = 0.99) or monocyte (P = 0.39) sub-populations.Neither GH Fig.  nor IGF-2− and H2O2, molecules whose creation precedes that of hROS [2− were significantly reduced in HCAECs following treatment at both physiological and supra-physiological concentrations of rhGH (333–3333 μg/L) and rIGF-1 (10–1000 μg/L). Using the non-specific ROS probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), Thum et al. [The principal finding of this study was that neither rhGH nor rIGF-1 exerted any significant effect on the rate of production of the highly reactive oxidants, OH(•) and ONOO-, under the respiratory conditions analysed, at any administered hormonal concentration. While this is the first study to examine the effects of rhGH/rIGF-1 on the production of these molecules, other studies have found that, in-vitro, these hormones exhibit significant effects on the generation of the reactive intermediates O of hROS , 32, 33. of hROS  found thm et al.  related 2− following only four hours of incubation. In addition, Thum et al. [m under the respiratory conditions analysed at any administrated hormonal concentration. The finding that neither hormone induced a change in the rate of hROS production is possibly due to the effect exerted on Δψm by saturating concentrations of respiratory substrates. In addition, saturating concentrations of ADP were also utilized in the study in order to initiate state 3 (phosphorylating) respiration following cellular permeabilization, which doesn’t accurately reflect endogenous respiration in-vivo where cells cycle between state 3 and state 4 (non-phosphorylating) respiration [Of note, the anti-oxidative effects reported in previous in-vitro studies of rhGH and rIGF-1 are the result of a 24–72 h treatment period , 33. In m et al.  also obsm et al. . In suppm et al. . In the piration . Thus, a2O2 generation, indicating that initial pro-oxidative effects preceded any changes in antioxidant capacity induced by the hormone. Together with the finding by Gustafsson et al. [Sanz et al.  found thn et al.  that rIGn et al. , 36. In n et al. . The pren et al. . GH defin et al. . SIRT4 in et al. . The expn et al. . In addin et al. . Such a n et al. .It is likely that modulation of oxidative phosphorylation by the GH/IGF-1 axis is not the only factor affecting the efficiency of electron transfer in-vivo. Other factors which induce oxidative stress by up-regulating the rate of mitochondrial ROS production likely contribute to an interactive effect on mitochondrial respiration which is necessary for the production of ROS at concentrations that will maximize their effect as intracellular messengers to induce an antioxidant response. Hence, a pre-existing pro-oxidative status within the cell may be necessary for GH/IGF-1 to induce any significant changes in antioxidant capacity, possibly explaining the absence of any significant effect on hROS levels, following pre-treatment with rhGH and rIGF-1 in-vitro in the present study.In animal studies, cellular oxidative responses to rhGH and rIGF-1 administration have varied depending on the cell type analysed, indicating that any antioxidant effects of the GH/IGF-1 pathway are tissue specific , 35, 40.The present study also found the level of mitochondrial hROS production to be significantly increased under conditions supporting NADH-linked respiration in comparison to all other respiratory conditions analysed, indicating that electron leakage at the site of complex I is an important contributor to the level of ROS production within the mitochondrial matrix. Such findings are in agreement with studies which have previously evaluated the rate of ROS production in isolated mitochondria , 47–49. 2 transferring electrons to ubiquinones on the inner mitochondrial membrane via ETF and ETF–QO [2− levels in isolated mitochondria from rat skeletal muscle and cardiac tissue when respiring on palmitoyl carnitine. The authors attributed this to a prolonged reduction of ETF and ETF-QO, in addition to an increase in complex I mediated electron leakage. Indeed, elevated concentrations of long-chain fatty acids in-vivo, in excess of the mitochondrial capacity to oxidize them, are known to become trapped in the mitochondrial matrix, elevating the redox state of the organelle and giving rise to the formation of highly reactive lipid peroxides [In addition, mitochondrial hROS production was significantly increased in the presence of octanoate in comparison to complex II linked respiration, indicating that reducing equivalents derived from β-oxidation play a role in the generation of hROS. It must be noted that β-oxidation generates equal amount of NADH, transferring electrons to complex I, and FADHd ETF–QO . Thus, td ETF–QO  demonstreroxides . Thus, leroxides .In conclusion, it is worth noting that high hROS values were recorded under saturated conditions of both complex I mediated and lipid-derived respiration which neither rhGH or rIGF-1 were capable of attenuating at the diverse hormonal concentrations tested. This could have direct implications for the administration of these hormones in-vivo. Indeed, GH has been demonstrated to induce substantial elevations in serum FFA availability in healthy subjects , 52, 53,1(5), 1,1′,3,3,3′,3′-hexamethylindodicarbo-cyanine iodide; EDTA, ethylenediaminetetraacetic acid; ETC., electron transport chain; ETF, electron transfer flavoprotein; ETF-QO, electron transfer flavoprotein quinine oxidoreductase; Fe2+, ferrous iron; FFA, free fatty acids; FoxO, forkhead box O; GH, growth hormone; GPX-1, glutathione peroxidase-1; H2DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate; H2O2, hydrogen peroxide; HBSS, hanks balanced salt solution; HCAECs, human coronary arterial endothelial cells; HPF, 3′-p-hydroxyphenyl fluorescein; hROS, highly reactive oxygen species; IGF-1, insulin-like growth factor-1; KH2PO4, potassium phosphate monobasic; MANOVA, multivariate analysis of variance; MAPK, mitogen-activated protein kinase; MgCl2, magnesium chloride; mIGF-1, local muscle specific IGF-1 isoform; Mn SOD, manganese-superoxide dismutase; MOPS, potassium morpholinopropane sulphonate; NO, nitric oxide; O2−, superoxide; OH(•), hydroxyl radical; ONOO-, peroxynitrate; PBMCs, peripheral blood mononuclear cells; PBS, phosphate buffered saline; PDK1, 3-phosphoinositide-dependent kinase; PI3K/Akt, phosphoinositide 3-kinase/protein kinase B; rhGH, recombinant human growth hormone; rIGF-1, recombinant insulin-like growth factor-1; ROS, reactive oxygen species; SDH, succinate dehydrogenase; SGK1, serum- and glucocorticoid-inducible kinase-1; Sir2, silent information regulator 2; UCP3, uncoupling protein 3; Δψm, mitochondrial membrane potentialADP, adenosine diphosphate; BMI, body mass index; BSA, bovine serum albumin; CCCP, carbonyl cyanide m-chlorophenylhydrazone; COX, cytochrome c oxidase; Cu, Zu SOD, copper, zinc-superoxide dismutase; DilC


About 14 cases have been described to date, but the vast majority of papers report exclusively conventional cytogenetic investigations and only two have been characterized by array-CGH.Ring chromosome 2 is a rare constitutional abnormality that generally occurs Here we describe the clinical, neuroradiological, and molecular features of a 5-year-old boy harbouring a ring chromosome 2 presenting with severe growth failure, facial and bone dysmorphisms, microcephaly, and renal malformation. Brain MR with diffusion tensor imaging revealed simplified cortical gyration, pontine hypoplasia, and abnormally thick posterior corpus callosum, suggesting an underlying axonal guidance defect. Cytogenetic investigations showed a karyotype with a ring chromosome 2 and FISH analysis with subtelomeric probes revealed the absence of signals on both arms. These results were confirmed by array-CGH showing terminal deletions on 2p25.3 (~439 kb) and 2q37.3 (~3.4 Mb).Our report describes a new patient with a ring chromosome 2 completely characterised by array-CGH providing additional information useful not only to study genotype-phenotype correlation but also to validate the role of already reported candidate genes and to suggest novel ones which could improve our understanding of the clinical features associated with ring chromosome 2.The online version of this article (doi:10.1186/s13039-015-0121-z) contains supplementary material, which is available to authorized users. Ring chromosomes result from one or two distal breaks followed by fusion of the broken ends and loss of the acentric fragments, but they can also be formed by fusion of subtelomeric sequences without apparent loss of genetic material. The ring chromosome phenotype is characterized by growth failure and intrauterine growth retardation (IUGR). According to Cote et al.  and KoszHere, we report on a boy with a ring chromosome 2 presenting growth failure, microcephaly, dysmorphic features, L-shaped kidney, and brain malformations studied with brain MRI and diffusion tensor imaging.rd centile), length 40.5 cm (<3rd centile) and head circumference 29.5 cm (<3rd centile). He developed mild respiratory distress requiring continuous positive airway pressure (nCPAP) for 72 hours. Echocardiogram depicted patent ductus arteriosus that disappeared spontaneously at follow-up. Abdominal ultrasound revealed crossed renal ectopia resulting in an L-shaped kidney (the left crossed kidney assumed a transverse position in the pelvis), while brain ultrasound showed incomplete opercularization.The patient is a 5-year-old boy, only child of healthy non-consanguineous parents. He was born to a 28-year-old mother whose pregnancy was complicated by IUGR and oligohydramnios, requiring caesarean section at 35 weeks of gestation. The Apgar score was 9 and 10 at 1 and 5 minutes, respectively. Physical examination at birth revealed left clubfoot  that was treated with a plaster cast. Birth weight was 1740 g  next to a minority of cells without the ring (8 metaphases). Figure A. The kaFAM110C, SH3YL1, ACP1, FAM150B). The deleted 2q37.3-qter region contains 27 OMIM genes  (NCBI build 37) and a large deletion of ~3.4 Mb  at the distal long arm of the ring chromosome at bands 2q37.3-qter  , while the deleted 2q37.3 region, in addition to the genes present in our deletion, contained the following OMIM genes: LRRFIP1, RAMP1, SCLY, HES6, PER2, TRAF3IP1, and ASB1  in all affected individuals, but ID is likely due to haploinsufficiency of multiple genes.The main phenotypic features, reviewed by Leroy et al. , includereported . The autKIF1A gene. KIF1A belongs to the kinesin 3 family, a large superfamily of molecular motors using microtubules as a “rail” to transport cargo along and chemical energy of ATP to drive conformational changes that generate motile force. Active transport of proteins along directional cytoskeletal filaments is fundamental for neuronal function and survival, because most of the proteins required in the axon and nerve terminals need to be transported from the cell body [KIF1A is implicated in mental retardation, autosomal dominant 9 (MIM 614255), neuropathy, hereditary sensory, type IIC (MIM 614213) and spastic paraplegia 30, autosomal recessive (MIM 610357).The deleted region in our patient contained also ell body . HomozygFARP2, encodes for a GTPase involved in neurite growth and axonal guidance. In particular, FARP2 is an immediate downstream signalling molecule of the Sema3A receptor complex that mediates repulsion of outgrowing axons and suppression of neuronal adhesion [Interestingly, our patient presented brain anomalies such as microcephaly with simplified cortical gyration associated with pontine hypoplasia, hippocampal malrotation and callosal abnormalities. In particular, the corpus callosum was dysmorphic with an abnormally thick splenium. DTI revealed the presence of a hypertrophic splenium suggesting an underlying axonal guidance defect altering the normal corpus callosum development. Intriguingly, another gene included in the deleted region of the present case, adhesion .TWIST2, NDUFA10, SEPT2, GAL3ST2, and NEU4, present in the deleted region, could have contributed to the overall phenotype of our patient.This peculiar combination of brain malformations has never been described in patients with ring chromosome 2 or 2q37.3 syndrome. This suggests that other pathogenic genes like FAM110C, SH3YL1, ACP1, FAM150B) did not appear to be causative of the phenotype of our patient, but we cannot exclude this hypothesis.Actually, the genes included in the deleted region 2pter-p25.3 . To better characterize the rearrangement, array-CGH analysis was performed using a 180 K platform with ~13 kb overall median probe spacing . Labelling and hybridization were performed following the protocols provided by the manufacturers. A graphical overview was obtained using the Agilent Genomic Workbench Lite Edition Software 6.5.0.18.Standard GTG banding was performed at a resolution of 400–550 bands on metaphase chromosomes from peripheral blood lymphocytes of the patient and his parents. FISH analysis was performed using Aquarius Subtelomere Specific Probe 2p (D2S2983) (Spectrum Green)/2q (D2S2986) (Spectrum Orange) . DTI data were acquired using a single-shot spin-echo echoplanar sequence in 32 directions with TR/TE: 9318 ms/71 ms, FOV: 224 × 224 × 120 mm, flip angle: 90°, 60 slices, acquisition voxel size: 2 × 2 × 2 mm, slice thickness: 2 mm, slice gap: 0 mm, 2 b values of 0 and 800. 3D/TFE volumetric T1-weighted images were also acquired for registration. DTI data were analysed and DTT was performed using the fiber tract tool supplied by the manufacturer .Written informed consent was obtained for publication and any accompanying images. A copy of the written consent is available.


Recently a few cases of synovial sarcoma (SS) of the abdominal viscera have been reported, raising awareness about the potential for confusion between this entity and KIT-negative gastrointestinal stromal tumors (GIST). We report the clinicopathological, immunophenotypical and molecular features of fifteen more SS occurring in the stomach (8 cases), epigastric region (one case), small intestine (one case), large intestine (three cases), involving both the terminal ileum and the caecum (one case) and liver (one case).SS18 gene region was screened in all cases: by conventional karyotype in one case, the remaining cases were screened either by interphase FISH or Q-PCR or both.Immunostains for SMA, DESMIN, CD34, CD117, S100, EMA, CK AE1/3, TLE1, CD56, CD99, BCL2, DOG1 were performed. Rearrangement of SS18 gene region rearrangement was demonstrated in all cases. A fusion transcript was amplified in eight cases: either SS18-SSX2 or SS18-SSX1 respectively in four cases each.Ten patients were male and five female, with an age range of 17–61 years (median 44). Tumor size ranged from 2 to 15 cm (median 8). Mitoses per 10 HPF ranged from 4 to 27 (median 9.5). Eleven tumors were monophasic fibrous SS, one biphasic SS and three poorly differentiated SS. SMA, Desmin, CD34, CD117 and S100 were negative in all cases, whereas EMA and/or CK AE1/AE3 were positive in all cases. TLE1, BCL2 and CD56 were positive in all tested cases. DOG1 was positive in one case. SS is increasingly recognized at visceral sites. Molecular analyses play a key role when dealing with usual histotypes in unusual sites. Correct diagnosis is crucial for appropriate therapy. Synovial sarcoma (SS) is a mesenchymal malignant tumour that accounts for approximately 10% of all soft tissue sarcomas . In apprSS18 gene (previously known as SYT) (on chromosome 18) with either SSX1, or SSX2 or SSX4 gene (on chromosome X) [SS subtypes share a common genetic alteration: a translocation involving chromosomes X and 18. This translocation results in three alternative fusion products of the osome X) .SS rarely occurs in unusual sites including: the head and neck region ,7, mediaFifteen cases of SS of the abdominal viscera were collected from three Italian institutions, the consultation files of one of the Authors (APDT) and Departments of Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA. Patients’ clinical records were retrieved. Follow-up information was available for 11 patients  Table . In a suImmunostaining was performed for EMA, cytokeratin AE1/AE3, SMA, Desmin, CD34, CD117, S-100, CD99, CD56 and TLE1. Four-μm sections of formalin-fixed paraffin-embedded material were used according to standard laboratory procedures. Details of the antibodies used are given in Table For case 14 conventional karyotype was performed. Cell culture, harvest conditions, and karyotyping were performed according to standard protocols.in situ hybridisation (FISH) was performed on 5 μm paraffin-embedded tissue sections using the LSI SYT (18q11.2) Dual Color Break Apart Rearrangement Probe set .Fluorescent Hybridisation was performed according to the manufacturer’s protocol. Slides were mounted and counterstained with anti-fade DAPI , visualized using an epifluorescent microscope (Olympus BX61) and analysed with FISH analysis software (Genetix-Cytovision 4.5.1). 300 interphase nuclei were analyzed.SSX and BETA2M genes, respectively. Samples were incubated at 42°C for 1 hour, then at 72°C for 15 minutes. PCR amplification of each sample and a 1:20 dilution were performed in duplicate using 96-well plates in 25 μl reaction mixture containing 300 nM of each primer, 200 nM of each probe (SSX1-SSX2) or 100 nM probe BETA2M and 2X TaqMan Gene Expression Master Mix , on Applied Biosystems StepOnePlus Real-Time PCR Systems (Applied Byosystem).  with the addition of proteinase K (Qiagen), and incubated for 72 hours at 55°C under moderate shaking. The percentage of tumor cells, as calculated from the HE-stained slides, was at least 70%. Subsequently, RNA was extracted with TRIzol-LS Reagent (Gibco BRL), according to the manufacturer’s instructions. RNA pellets were resuspended in 10–20 μl of RNAse-free water and stored at −80°C. 5 μg of total RNA were reverse transcribed in a total volume of 20 μl using specific reverse primers for In the identified fourteen cases of digestive system SS a male prevalence was found: male/female ratio was 3:1. Age at clinical presentation ranged from 17 to 61 years (median 44 years) ; one extended into the perivisceral soft tissue, peritoneum and omentum Figure A and twoMicroscopically, eleven of the tumours consisted of monotonous spindle cell proliferation, with scant intercellular eosinophilic collagenous stroma Figure B Table . A focalMitotic count ranged from 4 to 27 mitoses/10 HPF median 9.5)  karyotype (data not shown). The remaining cases were assessed either by interphase FISH   and epithelial markers (cytokeratins and EMA). Since morphological features of epithelial differentiation may be very subtle, immunostains are a valuable diagnostic aid. Cytokeratins tend to decorate most biphasic synovial sarcomas, but when dealing with the monophasic subtype, the percentage of immunopositivity falls to 60%-70%. Interestingly, cytokeratin immunoreactivity has been demonstrated only in 50% of PDSS  and highThe diagnosis of biphasic synovial sarcoma is usually straightforward, even for cases occurring in the digestive system. Regarding monophasic SS in the digestive system, the main differential diagnosis is with GIST: any mesenchymal lesion arising in the GI tract would naturally be suggestive of a diagnosis of GIST and CD117 negativity “per se” does not rule out such a possibility . GISTs aSarcomatoid carcinoma may also be considered in the differential diagnosis, however it often exhibits conspicuous pleomorphism, stronger expression of epithelial markers, and area of conventional carcinoma are often associated with the sarcomatoid component.EWSR1 gene [Stockman et al. proposed to call it malignant gastrointestinal neuroectodermal tumor [Gastrointestinal clear cell sarcoma may be very difficult to distinguish form digestive system SS ,34, alsoSR1 gene ,34. Remaal tumor .SSX18 or EWSR1 is crucial for differentiating respectively PDSS from Ewing Sarcoma/PNET [PD SS may resemble other small round cell tumors including Ewing Sarcoma/PNET, neuroblastoma, rhabdomyosarcoma and lymphoma . RemarkaSS18) and one of the synovial sarcoma X breakpoint (SSX) genes on chromosome X: SSX1, SSX2 and rarely with SSX4 [Cytogenetically, all SS variants are characterized by the reciprocal translocation t, which leads, at molecular level, to the fusion between the synovial sarcoma translocation gene on chromosome 18 (ith SSX4 -38. Inteith SSX4 . The SS1Despite initial attempt to correlate fusion type with a significantly longer disease-free survival ,40 morphRecognizing SS is of paramount importance to ensure the right therapy, especially because SS is known to respond to ifosfamide-based systemic treatments . In the